Christmas past Essay

The plot of the story goes as follows. Right from the start it is made clear that scrooge’s partner Marley is dead. Scrooge then goes several years without changing a single thing in the business. He and his only worker bob Cratchit work alone in the small dark office. Then one Christmas Eve two charity workers call round to ask him to make a donation. This is when we find out that scrooge despises charity. Later that day his nephew Fred comes to invite him round for Christmas dinner, an invite that is turned down by scrooge. That night when he gets home the ghost of his dead partener marley visits him. He tells scrooge that he must change his ways or he will end up like him not being able to allow his spirit to rest. But being force to â€Å"wear to chain he created in life. † Marley tells him that he is to be visited by three spirits over the next three nights. These spirits would show him the error of his ways. The first spirit is Christmas past. This spirit shows him his past again. The things he enjoyed as a young boy and his desisons he made that affected his current life. The next spirit is Christmas present. He shows scrooge what is currently happening in the people close to him and shows him what he is missing out on. The final spirit is Christmas future. He shows scrooge what will happen in his future if he doesn’t change his ways and that he will die a lonely old man. Scrooge then accepts the advice given by the spirits and generally becomes the ideal human often giving to charity and folks worse off than him. The first stave in the novello is when his former partner Marley visits scrooge. Right form the very beginning it is made clear Marley is dead. The first words on page one are â€Å"Marley was dead to begin with† â€Å"Scrooge new he was dead† as he signed for the register of his death. So when Marley appears to him his being there traumatizes him. Marley warns scrooge of the spiritual after life. He explains that man must share his humanity with others if he is not t be condemned to an after life. â€Å"It is required by every man†¦ that the spirit within him should walk abroad. † Scrooge also observes that Marley is â€Å"fettered† Marley explains â€Å"these are these the chains I forged in life†¦ I made it link by link, yard by yard of my own free will and of my own free will I wear it. † Marley then informs scrooge of what his afterlife will be like if he doesn’t change his ways â€Å"the weight and length of the chain you bare. † Marley then tells scrooge that he will be visit by three spirits, which will show him the error of his ways. Stave two tells the arrival of the first spirit the ghost of Christmas pass. This spirit, â€Å"a strange figure, like child like, old man viewed though some supernatural substance†, shows scrooge his most enjoyable moments of his past. One place which scrooge is taken is taken is his old work place, to Fezziwigs Christmas party. While there the spirit says â€Å"a small make to these silly folk so full of gratitude. † The spirit playing devils advocate by criticises Fessiwig causes scrooge to defend him â€Å"he has the power to flauntier† the spirit also shows him women which he once loved like his sister and a girl he once loved as a young boy. Stave three shows the next spirit, the ghost of Christmas present. This spirit, a large jolly old fellow, shows scrooge the true meaning of Christmas. First scrooge is taken to the cratchits house where he is shown how much such a large family appreciate so little. He has enough money to buy what ever he wants but then he realises that the simple things to him are luxuries to then that they can’t even really afford at Christmas, The joy on the faces of the children when they see that they are getting a pudding after dinner. He even is surprised to find that they are grateful to scrooge for putting the meal on the table even though scrooge knows that he is under paying Bob Cratchit. The spirit then takes scrooge to his nephew Fred’s home where he was having a Christmas party. Scrooge sees on the fun, which he is missing out on. The games and dancing which scrooge turns down as his despises sharing his fortune. Stave four shows the arrival of the final spirit, the ghost of Christmas future. This spirit, a ghostly figure dressed in black gown, takes scrooge to see his current future if he doesn’t change his ways. The spirit takes him to see his self in the future lay in bed. This is where scrooge comforts his own dead body without realising that he is dead. â€Å"Avarice, hard-dealing, griping cares? They have brought to a rich end truly. † â€Å"This is a fearful place†¦ In leaving it, I shall not leave its lesson, trust me and let me go! † scrooge finally realises what his dead body means: he loves his nerve and begs the spirit to let him go. The ghost insists, by pointing a finger that scrooge should lift the sheet to see the body scrooge responds â€Å"I understand you†¦ and I would do it if I could. But I have not the power, spirit. I have not the power†¦ † meaning that scrooge cannot bring him self to lift the sheet because it would mean coming to terms with his own death. Scrooge wants to kwon if anyone grieved for him â€Å"is there any person in the town who feels emotion cursed by this mans death, show me and that person spirit. † The spirit shows people who grieved for scrooge: ironically it is his debtors. When scrooge has died. They have lingered to pay off the debt. Scrooge is then taken to the churchyard to a churchyard: scrooge still needs to know the identity of the dead man but has lung suspected that it is himself. Scrooge is shown a tombstone and a new grave. Scrooge remarks ironically that the graveyard is a â€Å"worthy place† full of worthless dead bodies costumed by worms. â€Å"He advanced towards it trembling† he confronts the truth. Scrooge suddenly under stands the phantom:† he saw new meaning in its solemn shape†¦ † scrooge seems to understand that he can change his future. † Tell me I may sponge away the writing on this stone†¦ † scrooge is asking the spirit for a chance to avoid death. Scrooge is completely transformed â€Å"I will honour Christmas in my heart and try to keep it all the year. I will live in the past, present and future. The spirits all three shall strive within me. I will not shirk the lessons they teach. † The ghost fades away. Stave five is the final stave in the novello. It is when scrooge realises that he can alter his ways. â€Å"The time before him was his own, to make amends in†¦ † scrooge now seeks not to be a good businessman but to be a good man. â€Å"Glowing with his good intentions†¦ sobbing violently†¦ † yet even though scrooge has changed his way of life completely for the benefit of others. He is still happy with the new life he leads. â€Å"I am as a feather†¦ I am as happy as an angel; I am as merry as a schoolboy†¦ I am as giddy as a drunken man. † After scrooges complete change, the church bells ring out to celebrate Christmas morning and scrooge’s rebirth was on the birthday of Christ. Scrooge is completely changed: well-dressed and wearing a â€Å"delighted smile. † Scrooge rejoices in humanity: he enjoys the company of people and attending church. Now Scrooge visits Fred and accepts his invitation to Christmas lunch. Scrooge tells Bob Cratchit that he will raise his salary. Scrooge will become a â€Å"second farther† to tiny Tim. The transformation is complete. Scrooge has be come † as good a friend, as good a master, as good a man as the good old city knew†¦ â€Å"

Influential Leaders: Julius Caesar Vs. Mahatma Gandhi Essay

Influential Leaders: Julius Caesar vs. Mahatma Gandhi Julius Caesar and Mahatma Gandhi were both leaders from different countries and time periods. Based on the play, â€Å"Julius Caesar† by William Shakespeare, and the movie, â€Å"Mahatma Gandhi† directed by Richard Attenborough it is apparent that these two men were in some way very several similar and in other ways very different characteristics. Both were prominent people and lived lives of great importance and leadership. They both died in similar ways as well. But during their lives each man worked for completely different purposes. Julius Caesar and Mahatma Gandhi were both very influential people during their lives. Caesar was one of the top three men who had power in Rome. Many people respected and trusted Caesar as a leader. Caesar was even offered the crown many times but he refused it. Gandhi’s influence extended beyond the borders of India and reached out to the whole world. Gandhi’s philosophies inspired millions of people. Both men’s lives have ended in similar ways; they were assassinated. Julius Caesar was lured to the Roman Senate and murdered by several conspirators including his good friend Brutus. Gandhi was murdered by a Hindu fanatic who disagreed with his tolerance of Muslims. Caesar and Gandhi were both very courageous men. They stuck their necks out when they knew there were dangers out there. And the fact that they both were murdered shows how real those dangers actually were. Even though both Caesar and Gandhi were influential leaders, they each had very different views, ideas, and ways of going about doing things. Caesar was an important figure in his society and even had his own army to back him up. Caesar’s solutions to problems were solved by fighting, such as the time he fought Pompey and became victorious. Gandhi was different. He had no official political title, he did not command any army, and he did not amass any great wealth. Gandhi’s philosophy of non-violence incorporated civil resistance. He believed that non-violent civil resistance, not war, was the way to handle things. He also felt the only solution to hatred, ignorance and fear was love, truth and forgiveness. Caesar’s and Gandhi’s beliefs were quite different. Both men were assassinated in the middle of their lives, but for very different reasons. Caesar was killed because he was too powerful, and Gandhi was killed because he was too good. Caesar was too  powerful because he was a very ambitious man who was power mad. He even set himself up as dictator for life. Caesar wanted the power for himself, while Gandhi wanted the power for the people. Caesar was constantly living in fear of his life. He was superstitious and seemed on edge in several instances. Gandhi was thrown in jail and beaten numerous times and yet stayed persistent and determined despite all the unfair treatment he had suffered. Gandhi believed in Civil Rights and Democracy, which was the complete opposite of what Caesar wanted. Caesar always thought of himself as perfect and decisive. He loved to be in control and have all the power to himself. He enjoyed feeling higher and better than everyone else. He proved this by ignoring the warnings of the Soothsayer before arriving at the Senate. Gandhi was never an arrogant man as Caesar was. He always thought of himself as an equal, no better that anyone else. He dedicated his whole life to helping others. Gandhi exhibited his leadership by wearing homespun cloth that provided employment for poor people and revived the village economy. Gandhi was a very honorable man. Julius Caesar and Mahatma Gandhi were both very influential and important leaders. They both worked hard at the goals they hoped to achieve. Caesar and Gandhi shared a few character traits but also possessing several different traits, viewpoints, and ideas. Both men made impacts on history during their lifetimes and will be remembered for years to come as brave leaders who risked their lives to achieve their goals.

Older Adult Interview

I had the privilege of interviewing a 60 year old gentlemen who I will identify as Mr. E to protect his privacy for this assignment. The goal of my interview was to gain insight on aging from an older adult. I interviewed Mr. E in his home on a weekday evening. He expressed appreciation and was surprised that he was the focus of an interview in which his life story and thoughts would be recorded. Mr. E was born in a ranch in Guadalajara, Mexico. He is the youngest son of nine children. His father passed away when he was 1 – year old. He was privileged to attend elementary school from the 1st grade to the 4th grade.Mr. E had the responsibility of helping support the family as there were only two male children in the family and the remaining siblings were female. At 13 years of age he went to the neighboring state of Tepic, Nayarit to work in agriculture. He was 15- years old when he immigrated to the United States by himself. Mr. E lived with friends who helped him find a job 3 weeks after he moved to the United States. He worked as a busboy at a restaurant for 3 months. He left that job to work in the garment industry making jeans, shirts and blouses for 3 years. I was the only man working there at that time† (E. Privacy, personal communication, October 10, 2012). Mr. E observed that years later he saw more males seeking employment in the garment factories because word spread that any undocumented individual could work making clothing regardless of gender. He financially supported   two infant children on those wages. He then worked in a fabric for 7 years making electrical parts for cars. After that he worked as a gardener and left the business to his son when he retired. He was married at the age of 18 and had his first child at the age of 21.Two years later he had a daughter. He became a U. S. Citizen and has helped many family members also obtain their citizenships in the past decades. He is a grandfather of 5 and looks forward to seeing grea t grandchildren in the future. I asked Mr. E (2012) what he best enjoyed about being an older adult. You are a person that sees things for what they are. As if you walked a path and see what you could of done but didn’t. How could you have lived and not lived. You see your errors. Like when you are on a cliff looking down or on the clouds and looking down.When asked about challenges to getting older (2012) Mr. E felt that accepting the challenges and just living the best you can is all you can do. Try to live in peace and love what is on earth. When you think of death you have to accept it. Why fight it you are going in that direction. You have to make a decision. He told me a story of a friend he had who had cancer and she made the choice to stop the chemotherapy. Her arms had scabs and she decided enough was enough. She knew she wasn’t going to get better. She talked about death as if she were going to a party.He described how she appeared to be at peace because she lived a fulfilling life. Mr. E felt that she encouraged and motivated him more than he to her. Mr. E felt that the greatest joys of getting older were family and seeing it grow. He also felt that being loved and having others think highly of you were great achievements. Looking back on his life Mr. E felt that the only thing he could have done differently was to be more patient, smarter, more humane and not make as many mistakes. â€Å"You look back and think that you were not able to see things that are obvious† (E.Privacy, personal communication, October 10, 2012). When asked about fears of getting older Mr. E stated that living with diseases and not being able to pay for medications and hospitalizations was a concern for him. Although, he has insurance he stated it is very expensive and he is worried he might not always be able to pay the high amount. He stated that he worried about leaving family members behind that may not be emotionally and financially stable. ?The fina l thoughts Mr. E left me with were some positive things that he anticipate as getting older. Seeing the world as a paradise, enjoying spending time with horses and seeing family grow older and expand†(E. Privacy, personal communication, October 10, 2012). ?Throughout the interview themes such as family and time arose over and over again. His emphasis on missed opportunities with family has taught me that if I am not careful I will also have the same concerns when I am an older adult. He didn’t mention business as a regret even though when he talked about his personal history the majority of that conversation was on job history.During the interview I was on the edge of my seat because he had a lot of wisdom to share and I knew that I was lucky to get advice from somebody who has lived longer than I have. My perceptions of older adults has not changed as I have always felt that they have bigger wealth of information greater than Google. My new perceptions of aging are tha t healthcare is one of the biggest concerns for older adults. I need to hurry up and start planning for my own health care as I have not really given it priority in my life. This interview has confirmed my desire to work with older adults.

Should catholic priests be allowed to marry Essay

Should catholic priests be allowed to marry - Essay Example The vow of celibacy is not as simple as it sounds when the priests are taking it due to the temptations surrounding the world’s lifestyle and considering they are not cloistered religious men but mingle freely and interact with others. An interview of Fr. Sullivan by the New York Times yields that there are a section of Catholic Priests in various parts of the world who are married but still perform the same church duties as the rest of the celibate priests without any hindrance. Their wives assist them perform God’s work not hinder them (Oppenheimer 1). There has been a recent decrease in the number of ordained Catholic priests as the number of the Christians faithful continues to increase. According to the cbs news (Quijano), this shortage in the catholic priests has even necessitated the Roman Catholic Church to take up what is seemed as â€Å"unorthodox methods† of seeking former Anglican priests (who are married and with children and do not subscribe to the Roman Catholic Church doctrines) to fill up the blank positions. Statistics indicate that the number of Catholic priests has reduced from approximately 59,000 in 1975 to around 39,000 as of 2012. This is relation to the increase in number of followers by approximately 17million between 1975 and 2012. These statistics are only for United States of America and not in other countries all over the world. According to critics, this shortage is as a result of the celibacy vow which even though people want to become priests and serve God, the vow restricts them from marrying whereas many of them want to have their own family to continue their family lineage. Allowing marriage for the celibate priests will prevent and even tone down some of the â€Å"ungodly† behavior always being aired in the media including claims of sodomy with young boys, having multiple female sexual partners with some having their babies (which always is done in the dark). This

Critical Thinking Assignment Example | Topics and Well Written Essays - 1000 words - 1

Critical Thinking - Assignment Example The key question that was asked was very simple: Why? As the commission said in the report, â€Å"Why did they do this? How was the attack planned and conceived? How did the U.S. Government fail to anticipate and prevent it? What can we do in the future to prevent similar acts of terrorism?† Much like the public on September 11, 2001, the parties involved in this report were also in a state of shock, asking themselves how and why such a thing could come to pass on American soil. Much of what the committee needed to put together their report was already in evidence, gathered by various agencies as soon as possible after the actual events of September 11, 2001. The committee in the report was very mindful that they were writing â€Å"with the benefit and handicap of hindsight†, meaning that in looking back, there were various events and actions, such as â€Å"not discovering false statements on visa applications, or not recognizing passports manipulated in a fraudulent manner†, as well as reports delivered to the United States government from around the world, that had they been linked together in any way would have probably led them to realize that the real threat of the attacks did not lay abroad in another part of the world, as they had originally thought, but right on American soil. One of the biggest concerns of the 9/11 Commission was the lack of centralized response system to the emergency situation once the attacks were underway. The report specifically states that â€Å"the defense of the U.S. airspace depended on close interaction between two Federal agencies: the Federal Aviation Administration (FAA), and the North American Aerospace Defense Command (NORAD).† However, there were fundamental problems even in the communication between the two agencies, as there was no protocol in place or procedure to follow in the case of a hijacked airplane being used as a weapon,

Communication Challenges to Managers in Global Virtual Teams Research Paper

Communication Challenges to Managers in Global Virtual Teams - Research Paper Example It is therefore important to study and understand the connection between communication and performance of virtual teams. Jointly, trust and communication vastly influence performance of members of virtual teams. In any kind of team, members are likely to differ in ideologies as well as ambitions, which can be challenging to managers managing such teams. In global virtue teams, the challenges are severe since the teams comprise of members from different nationalities characterized by cultural as well as technological differences. Being a leader of a global virtual team is challenging and it requires excellent management skills. This paper focuses on communication as one of the crucial challenges for managers in global virtual teams, because of differences in their national culture and technology structure. Challenges posed by geographical separation include lack of synergy. Another challenge that managers of global virtual teams face is inability to identify the talents of their employees thus might end up employing a less diverse team. Virtual teams permit organizations to expand their territories thus employ individuals from different backgrounds. However, challenges may arise in the management of virtual teams resulting in ineffectiveness (Mirjaliisa, 2007; Sarker, Ajuja, Sarker, & Kirkeby, 2011). The challenges could be related to several factors. Leadership style is the major challenge. Managers should thus ensure that their leading strategies are in line with the team members’ anticipation. This can be made possible by switching between the different leadership styles depending on the situation at hand (Johri, 2010; Shachaf, & Hara, 2007; Shachaf, 2008; Dorothy, Kayworth, & Mora-Tavarez, 2010; Karen, 2008). The other challenge is the building of trust. The geographical separation often results in a feeling of alienation, which makes it hard to develop strong relationships amongst team members. The lack

Apple Store Research Paper Example | Topics and Well Written Essays - 500 words

Apple Store - Research Paper Example The Pasadena Apple store has a variety of products and services. The store has a variety of products and services that meets all age groups and both male and female customers. The store deals in hardware products like iPod touch, iPhone, Apple Cinema Displays, Apple TV, Mac Pro, Mac Mini and Airport Cards (Carlton 165). They have a variety of Apple accessories like mice and wireless keyboards. They have up to date online services termed as Apple Store online. Customers can configure their phones for fast and efficient services. The store has products like computers, which has faster and larger hard drives than any other computer store. The store has simplified inventory that provide instant availability of information on the products and services (Jeffreys 254). The store provides its customers with Apple software that include IWork and ILife applications bundles, Mac OS X, Shake, DVD Pro Studio and miscellaneous software titles. The store has games, quality printers, memory upgrade software, scanners and digital cameras. The store has up to date brands like Mac OS X and Mac Book. Apple Inc has designed this store to suit customer’s needs (Jeffreys 252). The store provides Mac Book brands that meet the customers’ requirements. The store is in line with the California’s regulatory authority. The store has product licenses that are verified by the local authority and the Apple Inc to provide efficient services to their customers (Terry 4B). The retail shop has evolved from just being a computer company to a true consumer brand. The company initially had Mac Book computer line, however, after the expansion; the retail shop added the Iphone and IPod (Lewis 121). This has made the retail shop be ranked one of the best in California. The overall interest of the quantity and product assortment of the retail has improved over the years. The retail shop is producing a variety of products meeting their

Amazon case study Essay Example | Topics and Well Written Essays - 750 words

Amazon case study - Essay Example evolution includes change of ownership as everyone is incorporated since the owners decides issues based on the long terms motivated by the excess opportunities available in the modern world due to development in technology, labor quality increase over the times since hiring and placement must be done to acquire competent efforts who can pursue diversification in all aspects. Companies diversify in different ways. Some uses the same line of products to produce more known as the related diversifies while others chose to go different line known as the unrelated diversifiers. Thus those who chose to be in the same line tends to make more profit than the firms that deviate to other goods since the companies diversifying in similar products tends to specialize and produce quality goods which can compete relatively in prices at the market. Amazon is a company that is customer obsesses ion and tries to satisfy them by all means since they are the key to the growth of the firm hence participates in various inventions to improve productivity (KENNY, 200). The organization also believes in spending the right way to achieve self-efficiency of the diversification, increase sales volume and become more resourceful and innovations to all sectors in the company. Amazon chose to diversify into unrelated business so as to expand their growth and the evolution as gone over times. The company was started as an online bookstore selling but that has since changed over the times. The company has now incorporated diversity in other areas which includes, software’s, MP3s, video games electronics, food to consumers, music CDs, video tapes, equipment, fashion and many more. The company also has got a multi-product strategy in the market as it plans to enter the sale of air tickets online and hotel rooms. The company has since acquired a substantial share of the market of the bookstore. However as a result of the diversification, the sales volume has greatly reduced over the times

The Exchange Rate Regime of Thailand, purchasing power parity of Essay

The Exchange Rate Regime of Thailand, purchasing power parity of Thailand - Essay Example Population of Thailand is relatively homogeneous, which consists of Buddhist 94-95%, Muslim 4-5%, Christians, Hindus and others. More than 85% of its population speak Dialect of Thai and share common culture. Like many other countries of the world, Thailand also witnessed many ups and down and was occupied by the Japanese during Second World War. Since Japan’s defeat in 1945, Thailand has had very close relations with the United States. Threatened by communist revolution in neighboring countries, such as Vietnam, Cambodia and Laos, Thailand actively sought U.S assistance to contain communist expansion in the reason. Recently, Thailand also has been an active member in multilateral organizations like the Association of South East Asian Nations (ASEAN) and the Asia-Pacific Economic Cooperation (APEC) forum. 1.1 Economic performance of Thailand: - The Thai economy is export dependent, with export accounting for 60% of GDP. Thailand recovery form the 1997-98 Asian financial crises relied largely on external demand from the United States and other foreign markets. The Thaksin government took office in February 2001 with the intention of stimulating domestic demand and reducing Thailand reliance on foreign trade and investment. Since then Thailand has embraced a â€Å"duel track† economic policy that combines domestic stimulus with Thailand’s traditional promotion of open market and foreign investment. Weak export demand held 2001 GDP growth to 2.1%. Beginning in 2002, however, domestic stimulus and export revival fueled a better performance, with real GDP growth at 6.9% in 2003 and 6.1% in 2004. Before the financial crisis, the Thai economy had years of manufacturing-led economic growth –averaging 9.4% for the decade up to 1996. Relatively abundant and inexpensive labor and natural resources, fiscal conservatism, open foreign investment policies, and encouragement of the private sector underlay the economic success in the years up to 1997. The economy is

Whole Food Market, USA Essay Example | Topics and Well Written Essays - 2500 words

Whole Food Market, USA - Essay Example The objective of this study is to outline marketing mix and market entry strategy for Whole Food Market, Inc. while entering into Netherlands. Cultural dimensions of new market would also be included in the study so as to reflect upon its impact on proposed marketing mix. The entire study shall be centered towards global market strategy of Whole Food Market, Inc. 3. Main Findings 3.2 Marketing Mix of Whole Foods Market, Inc., Global Expansion 3.2.1 Product ïÆ'Ëœ Concept: Organic products and natural food products would be delivered in Netherlands market. ïÆ'Ëœ Whole Foods Market, Inc., Global Strategy: Global strategy shall be either direct exporting or wholly owned subsidiary. ïÆ'Ëœ Brief Adaptation or Standardization in Netherlands: Standardization product strategy would be applied in Netherlands market in order to sustain their competitive advantage (De Burca, Brown and Fletcher, 2004). 3.2.2 Price ïÆ'Ëœ Concept: The main concept is to acquire maximum market share and establish a stable market position. ïÆ'Ëœ Whole Foods Market, Inc., Global Strategy: In global context, the focus would be on market development strategy. ïÆ'Ëœ Brief Adaptation or Standardization in Netherlands: Adaptation approach will be applied on Netherlands market in the context of pricing strategy. 3.2.3 Place ïÆ'Ëœ Concept: Marketing mix element place is related to distribution network. Whole Foods Market will be delivering products to manufacturing or processing plant to the warehouse and finally to retail stores.

Project Management Essay Example | Topics and Well Written Essays - 3500 words

Project Management - Essay Example All these factors are not driven on product and service quality but, also on how to achieve them, so it does not entails the latter two but also quality assurance and control of the process in addition to the end product for an overall even and good quality. Contrary to the other software development, which can be termed in many different ways good examples, are software application development, software design, platform development, and many others. However, all said it is the development of a software product, they may include research in development of new designs, photo typing, reuse modification, maintenance, and re-engineering of result oriented software products. By trying to define it, we can say it is a structure driven on development of a software product. Then in trying to understand the two first, let us look at the former software development projects. There are different approaches of software development. Nevertheless, all this approaches share a common understanding a nd towards the following laid down processes: analysis of the problem, a market research on the problem, coming up with requirements for the proposed business solution. There is also generating a planned design for the solution based on the software, implementation of the software, a test drive for the software, use of the software in the market and lastly maintenance and fixing of any abnormalities in its use (Brooks 2005). Software development project are projects just like any other and to relate them to quality management one has to simplify the and try to understand them that way as to have a clear understanding of the two of them .quality management can be a big element too a smooth running of a software development project. Through quality management, a software development project is derived. for one to come up with a workable and profitable project one has to use and implement the workability’s and elements of quality management for this reasons we have to look at th e ways and elements of quality management to understand the similarities, success and failures of software development projects as it is as any other project in quality management. There are certain elements that are adhered to in quality management that are essential and vital to project control these are; organization structure, responsibilities, data management, processes including purchasing’ resources natural and human resources, customer satisfaction, continuous improvement, product quality, maintenances, sustainability and transparency. All this factor has to be incorporated in system development project for it is to be viable (Brooks 2005). For a viable system, quality system adhere to certain elements that are co related and brings out a good relationship between the two that is quality management and system development success these are personnel training and qualification; control of product design, documentation, product design and its purchasing power, product id entification, traceability at all stages of production. Process of controlling and defining the production both the systems and the product at the same time in this scenario. The production software, inspection should be defined and controlled and ensuring the test equipments is measured to standard. There is need for process validation, acceptance of products, reduction, and control of

Us Womens History Essay Example for Free

Us Womens History Essay The Native American women were trained to work hard in the fields and in the house. They were held responsible for over 75% of food production and the gathering of the fruits in the community. These women were responsible for making clothes for themselves and their families from the skin of rabbit and dear. The Native American women were allowed to dress in long dresses and leggings. The Native American women mostly practice agriculture when civilization of Europeans arrived and because of all their lives depends agriculture their economy grows with their own hard work spiritual role. As the women are the backbone of the community, they are given right to own land and in this they use for farming and inherit it to their descendants. In America native communities in 1600 century, women are given to have much power than European women, this is because Native American women are very hard working and they also have good feelings and near to their husbands, thus they are given equal opportunity to rest of the community. Status of Women in the Southern and Northern colonies In the southern colonies women were legally subordinate to men, politically and nonetheless improved economically in the colonial period. Southern colonies the women were view less than the men, where they took advantage for their right in the society of inheriting the land of their late husbands. The southern colonies focus was on profit while northern colonies focus on religion. The northern colonies are better in status than southern colonies because northern women are married earlier, they had larger families and they live longer than their cousins on the other side of the ocean. Living conditions in the early Northern and southern colonies Living condition in early 1600 in north and south, colonies used Americans as slaves and servants for their plantations, but in late 1600 the African slaves became the primary source of American slaves. Southern plantation used to give huge profit to the northern merchants. Colonies came to America for religion and looking for job as most of them escape war. Although they got a lot of resources, but they started colonizing Americans living condition of Americans was too poor and white colonizes took these advantage and started employing some of the Americans and some of Americans bought as servant with 25- 50 dollars. The servants were given a small grubstake and if she or he was lucky, a few acres of land. Thus, some of the servants were treated fairly. Servants or slaves who are living at southern were treated as slaves without any payment. (Kramarde, Cheris. and Spender, Dale. 107) Work cited Kramarde, Cheris and Spender, Dale. Routledge International Encyclopedia of women. Routledge. (2000).

Human Carbonic Anhydrase II

Human Carbonic Anhydrase II Human carbonic anhydrase II is one of the fastest studied enzymes known with a variety of roles in reaction catalysis. Its primary function is to catalyze the reversible hydration reaction of carbon dioxide. In addition to carbon dioxide hydration, it is also capable of other latent skills, such as catalyzing esterase activity. The ability of human carbonic anhydrase II to function as a catalyst derives from key residues in and around the active site that play crucial roles in the mechanism. Substitutions to two of those particular key amino acids were performed via Quick-change site directed mutagenesis: H64A and V142D, to investigate the particular role they have in the catalytic active site. Various kinetic experiments and structural analyses were performed on wild-type carbonic anhydrase and the mutants to discern and compare their activity to each other and to literature, including Michaelis-Menten parameters for PNPA hydrolysis, CO2 hydration, and inferring function molecular m odelling. Though the same trends can be seen as the literature, individual values were found to be much lower owing to errors in measurement and equipment. Trends were found to coincide with the mutants known roles in the active site: His64 is the proton shuttle that facilitates proton transfer during the rate limiting step and Val142 participates in the hydrophobic pocket to bind and recruit substrates to interact with the active site. Mutations to both of these sites show that enzyme efficiency and activity strongly decreases. Introduction Human carbonic anhydrase II (hCAII) is a zinc metalloenzyme that catalyzes the following reversible reaction: . The enzyme commonly functions to help shuttle carbon dioxide in red blood cells to rid the body of metabolic waste, and catalyzes the hydrolysis of many aromatic esters [1, 2]. Structurally, a zinc ion is located in the active site, coordinated to 3 histidine residues (H94, H96, H119) and usually a hydroxide ion or water molecule [2]. The mechanism of hCAII proceeds through two major steps: 1) the conversion of carbon dioxide to bicarbonate, and 2) the regeneration of Zn-OH by proton transfer. The active hydroxide that is bound to zinc nucleophilically attacks a nearby carbon dioxide molecule, resulting in a bicarbonate ion binding to zinc [3]. The zinc-oxygen bond breaks to subsequently release a bicarbonate ion, which is replaced with water [3]. The Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH bond is regenerated by a proton transfer to the external buffer, which is facilitated by the His64 residue [3]. The proton transfer step is the rate limiting step of the reaction [3]. The diazole side chain on the histidine residue is what gives it the ability to be a proton acceptor and donor. Mutations in that position (His64) usually result in decreased enzyme activity due to a lack of proton transfer; however the reaction does proceed to a lesser degree without an active His residue, possibly due to its extensive water network in the activ e site forming secondary proton wires [4]. Carbonic anhydrase catalyzes one of the most rapid reactions; it is one of the fastest enzymes studied [1]. Its reaction speed is due, in part, by the amphiphilic nature of the active site [1]. The hydrophobic side is used to bind carbon dioxide, while the hydrophilic patch functions to optimally orient the carbon dioxide molecule for the reaction [1]. The hydrophobic wall forms a well-defined pocket near the zinc-hydroxide and is composed of the following amino acids: Val142, Val121, Leu197 and Trp208. The hydrophilic patch consists of Thr198 and Glu106, which form a hydrogen bond network with the Znà ¢Ã¢â€š ¬Ã¢â‚¬â„¢OH to stabilize and orient it for nucleophilic attack on CO2 [2]. Therefore, any modifications to the hydrophobic pocket would change its structure, and consequently, its catalytic efficiency [1]. In this study, the importance and role of His64 and Val142 to the structure and mechanism of hCAII are determined through site-mutagenesis and subsequent characterization of the new mutants, H64A (His64 Æ’Â   Ala) and V142D (Val142 Æ’Â   Asp) via kinetic and structural analysis. The changes that arise from the substitutions may prove to be applicable to drug synthesis because hCAII is known to be involved in a variety of diseases, for example, Marble brain disease, where mutations in the hCAII gene leads to a deficiency in the enzyme which is an autosomal recessive disease [5]. Studies in hCAII mutations can be used to design folding modulators to suppress misfolding which frequently occurs due to hCAII destabilization [5]. Another major disease involved with hCAII gene is osteopetrosis. The hCAII genes inactivation decreases osteoclast function in bone, and knowledge of hCAII mutations that inactivate the enzyme may lead to better understanding of bone remodelling [6]. Some carbonic anhydrase diseases use inhibitors (CAI) to suppress the hCAII as a therapeutic treatment. Inhibitors prevent hCAII activity by inhibiting either of the reaction steps: the conversion of CO2 which involves V142 in the hydrophobic pocket, or the rate limiting step, proton transfer, in which His64 is crucial. Experimental Procedure Site directed mutagenesis via the PCR-based Quick-change method was performed on hCAII as cited in Woolley (2011) for 10 ng and 20 ng wild-type plasmids (hCA2pET24b from Novagen) [7]. Table shows the sequence of the primers used in the PCR reactions. Products of PCR mutagenesis reactions were run on 0.7% agrose gels to determine size. The gels were run at 150 V in 1X TAE buffer. Red safe dye from Intron Biotechnology was used in the agrose gel instead of ethidium bromide for safety reasons [7]. The standard molecular weight ruler used was a 1 kB DNA ladder from Fermentas. Table : Primer sequences used in mutagenesis of hCAII in the forward and reverse direction for mutants H64A and V142D Mutant Direction Sequence MW (Da) %GC TM ( °C) H64A Forward GGATCCTCAACAATGGTgcTGCTTTCAACGTGGAG 10778 51 67 Reverse CTCCACGTTGAAAGCAgcACCATTGTTGAGGATCC 10709 V142D Forward CTGATGGACTGGCCGaTCTAGGTATTTTTTTG 9868 44 62 Reverse CAAAAAAATACCTAGAtCGGCCAGTCCATCAG 9779 The enzyme, DpnI, was then used to digest methylated DNA (the parent template DNA). The DNA vector that contained the mutation was transformed into supercompetent E.coli turbo cells from New England Biolabs by heat shock [7]. LB-agar plates were prepared to grow the transformed cells containing mutant genes (i.e. H64A and V142D hCAII gene) [7]. Both were injected with Kanamycin to ensure that the culture that grows will have the desired mutation [7]. A miniprep culture was set-up from the LB-agar plate into LB medium to grow one colony for DNA analysis [7]. Restriction enzyme mapping was prepared and XhoI and BglII were chosen, they were used under buffer 3 for optimal efficiency. Plasmid putification was performed using the QIAprep Spin Miniprep Kit, and then the chosen restriction enzymes were carried out and were run on 1% agrose gel [7]. A sample of the purified DNA was sent to an external company (ACGT) for commercial sequencing (Sanger dideoxy type) to verify if the mutagenesis occurred correctly. The sequence was analyzed using the program BioEdit. To determine the level of confidence of the sequencing results, the purified DNA was quantified using UV/Vis absorption via a spectrometer [7]. The concentration was calculated using Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1(260) as 50 ÃŽÂ ¼g/mL. Purified plasmid DNA was transformed into E.coli BL2(DE3) cells to initiate protein expression by heat shock, similar to the transformation into turbo cells [7]. The cells were cultured and a single colony was grown. Once sufficiently grown, ITPG and ZnSO4 were added to induce protein expression [7]. SDS-PAGE was used to confirm protein expression and was analyzed against an unstained protein molecular weight marker by Fermentas. The protein and ladder was stained with coomassie blue [7]. Affinity chromatography was used to purify the mutant hCAII proteins [7]. The matrix used was agrose linked to p-(aminomethyl)benzenesulfonamide, exploiting the tight binding that occurs between hCAII and sulphonamides. Once purified, the protein was dialyzed using a 6000-8000 Da dialysis membrane to replace the elution buffer with protein buffer and removes the matrix from the protein [7]. SDS-PAGE is again used to confirm the protein is still present after purification and to check its approximate molecular weight. It was run for two different amounts of protein, 2 ÃŽÂ ¼g and 10 ÃŽÂ ¼g, and also ran 10ÃŽÂ ¼L of wash fractions from affinity chromatography [7]. Protein concentration was determined by UV absorption at 280 nm in a final concentration of 6M guanidine hydrochloride. From the calculated concentrations, purity of the protein could be assessed via SDS-PAGE. To characterize this purified hCAII protein, a variety of analyses were done. Two types of mass spectrometry (MS) were performed: electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) [7]. The MS analysis was used to confirm the presence of the mutation in hCAII with intact and digested protein. Protein samples (H64A and V142D hCAII) were not diluted for either of the MS analyses as cited in Woolley (2010). Samples of 10 ÃŽÂ ¼L of stock protein concentrations (37.6 ÃŽÂ ¼M H64A and 3.2 ÃŽÂ ¼M V142D hCAII) were used for analysis of the molecular weight of the intact protein by ESI-MS. Both mutants were then digested by Trypsin Gold (MS grade) from Promega and the resulting fragments were evaluated by ESI-MS as well [7]. A 50 ÃŽÂ ¼L sample was used for each mutant, 40 ÃŽÂ ¼L of the mutant at stock concentration and 10 ÃŽÂ ¼L of the Trypsin Gold. A couple ÃŽÂ ¼L of the digested mutants were saved for MALDI-MS and the rest was used for ESI-MS. Once the molecular weights for each of the digested fragments were determined by ESI-MS, the products were run through a protein database to confirm the identity of the protein and mutations [7]. The 1 ÃŽÂ ¼L of the tryptically digested mutants prepared for ESI-MS, subsequently underwent MALDI-MS. The 1 ÃŽÂ ¼L samples were mixed with a matrix consisting of 1 ÃŽÂ ¼L ÃŽÂ ±-cyano-4-hydroxycinnamic acid (CHCA) and 1 ÃŽÂ ¼L of 0.1% trifluoroacetic acid (TFA) [7]. The entire mixture was pipette onto a MALDI well and was inserted into the mass spectrometer and a MALDI-MS spectra was obtained. Michaelis-Menten kinetics was used to determine the KM and kcat of the p-nitrophenyl (PNPA) hydrolysis reaction [7]. The ionized product from the hydrolysis, p-nitrophenol (PNP) produces a bright yellow colour that was used to follow the rate of the reaction via the Perkin Elmer Lambda UV/Vis spectrophotometer [7]. Various sample concentrations of PNPA were set up to have a final enzyme concentration of 0.2 ÃŽÂ ¼M in protein buffer [7]. The initial rate measurements of each PNPA concentration were taken for wild-type enzyme, H64A mutant, V142D mutant, and a blank with no additional enzyme added (refer to data tables in Enzyme Kinetics I [7]). PNP has a molar absorption coefficient (ÃŽÂ µ) of 1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1. This was used to calculate Michaelis-Menten values: Vmax, KM, kcat, and kcat/KM [7]. The ability of hCAII mutants (H64A and V142D) and wild-type hCAII to catalyze the hydration of CO2 was measured. The pH of the solution was measured to track the progress of the reaction because the reaction generates protons. Enzyme solutions were prepared according to table 2 in [7]. The buffer used in the table was 50 mM TRIS buffer (pH 7.8). Additional enzyme samples were prepared for 25 nM of wild-type hCAII and 100 nM of H64A mutant in a final concentration of 22.5 and 29.92 mM imidazole buffer (pH 7.8) respectively to determine chemical rescue of mutant H64A. The pH of the CO2 hydration assay was measured using a pH probe and pH meter at 5 second increments for a total of 90 seconds starting at the beginning of the reaction [7]. The slope of the initial changes in the first 2 points was considered to be the V0 for each enzyme concentration. From the initial velocity, a kcat value can be calculated for each enzyme using the assumption that [S] >> KM, the Michaelis-Menten equati on simplifies to kcat=V0/[E]. The third kinetics experiment used fluorescence to determine the binding constant of dansyl amide (DNSA) and acetazolamide (AZ) (from Sigma-Aldrich) to H64A and wild-type hCAII was performed using the Perkin Elmer Fluorometer [7]. Stocks of 1 mM and 200 ÃŽÂ ¼M of DNSA were prepared from a 21.6 mM DNSA stock by dilution with DMSO. Enzyme stocks were diluted to 0.25 ÃŽÂ ¼M with TRIS buffer to make a 10 mL solution. A 1 mL sample of H64A from stock made was titrated with DNSA in small increments [7]. The fluorometer emissions were taken at 470 nm. AZ titration in competition with DNSA was not able to be completed. The last characterization experiment done was molecularly modelling the hCAII wild-type enzyme, as well as the mutants H64A and V142D. The molecular model of hCAII analyzed was derived by x-ray crystallography and found in the Protein Data Bank (PDB) repository. The wild-type and H64A hCAII structures examined had a PDB code of 1CA2 and 1MOO respectively. At present, no crystal structure has been found for V142D hCAII. The Swiss PDB Viewer program was used to visualize the protein structures. Secondary structures of the proteins were able to be observed. Residues around the metal active site and the Ramachandran plot were explored. Homology between hCAII and other carbonic anhydrase isozymes, hCAIV (PDB code 1ZNC) and hCAI (PBD code H1CB), were also studied by performing an iterative magic fit on the ÃŽÂ ±-carbons and structure alignment for each pair. The root mean square (RMS) between hCAII and the other isozymes were also analyzed to determine conserved and deviated regions in the structures. The binding of cobalt in the hCAII active site was also investigated (PDB code 3KOI). The structural inhibition of hCAII by AZ was also gleaned by structural analysis (PDB code 3HS4). Its mode of inhibition and binding sites were shown through the crystal structure. Lastly, the Swiss PDB Viewer program was used as a tool to theoretically synthesize mutations and compare it to the actual structure as determined by other scientists, for example, by aligning the virtual and crystallized mutations to determine deviations in structure by performing RMS. Results Site-directed mutagenesis PCR. Products from the PCR mutagenesis reactions were examined using 0.7% agrose gel electrophoresis. Two samples of differing amounts of template DNA (10 ng and 20 ng) were used for each mutant (Figure ). Bands were only observed for samples containing 20 ng of the hCA2pET24b DNA template plasmid (Figure ). The size of the bands observed coincides with the size of the plasmid used, 6018 bp. Heat shock transformation and isolation of plasmid. Several colonies were observed after plasmid transformation for both mutants, and 1 colony from each mutant was chosen for restriction enzyme digest with BglII and XhoI. Figure : Electrophoretic run on 0.7% agrose gel of DNA of hCAII mutants from PCR mutagenesis reactions. Lane 1 is the GeneRuler ladder by Fermentas and lanes 10-13 are the following: V142D (10 ng), V142D (20 ng), H64A (10 ng), and H64A (20 ng). As suggested from the gel, the mutants in the 20 ng plasmid was more successful than the 10 ng plasmids in determining relative molecular weights. Both mutants in the 20 ng plasmid show a band at approximately the 6000 base pair mark, which coincides with the number of base pairs in the hCA2pET24b plasmid that was used (6018 base pairs). Quantification of pure plasmid DNA. A 1/20th dilution was carried out on the purified DNA with elution buffer (EB; 0.1 M Tris, 0.4 M KSCN, pH 7). The absorption of the diluted DNA at 260 nm and 280 nm was taken by a UV/Vis spectrophotometer and the relative DNA purity was determined (Table ). The assumption that Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL for DNA was applied in the calculation of concentrated and diluted concentrations of purified DNA (Table ). Table : Relative DNA purity for mutants V142D and H64A determined by UV/Vis spectrophotometer absorbance at 260 and 280 nm. Calculated concentrations of mutants from absorbance data, where Ecà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 260 = 50 ÃŽÂ ¼g/mL. Mutant Wavelength, ÃŽÂ » Absorbance Units Relative DNA Purity (A260/A280) Concentrated (ÃŽÂ ¼g/mL) Diluted (ÃŽÂ ¼g/mL) V142D 260 nm 0.3117 1.7852 311.70 15.59 280 nm 0.1746 H64A 260 nm 0.2653 1.7581 265.30 13.27 280 nm 0.1509 Enzyme restriction digest. Purified plasmid DNA of mutants were digested with XhoI and BglII, separately and together in a single and double digest for both mutants. The digested and undigested samples were run on 1% agrose gel, and 2 bands were observed around the 6000 and 7000 bp marker for all 8 samples (Figure , Figure ). The expected length of the bands in the double digest should be 892 bp and 5126 bp (Figure ). Figure : Electrophoresis performed in 1% agrose gel of digested V142D hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: V142D plasmid, V142D + XhoI, V142D + BglII + XhoI, and V142D BglII. Double bands are shown at the 6000 and 7000 bp marker for all 4 V142D samples. Figure : Electrophoresis performed in 1% agrose gel of digested H64A hCAII in lane 1-4. The (1 kB) GeneRuler DNA ladder is shown in lane 5. Lane 1-4 contain the following: H64A plasmid, H64A + XhoI, H64A + BglII + XhoI, and H64A BglII. Double bands are shown at the 6000 and75000 bp marker for all 4 H64A samples. Figure : Restriction enzyme cut sites and position of hCAII gene (5072-5854) on the hCAI2pET24b plasmid DNA Sequencing. The mutations for both V142D and H64A in the hCAII gene were successful according to the sequenced DNA result obtained from ACGT. Other mutations in the DNA sequence were observed in both mutants, but since the aligned protein sequence was the same, mutations were likely to be silent mutations due to amino acid redundancies. When sequenced in the forward direction by T7 polymerase, a protein mutation was found (K153N) other than the desired mutation of V142D; however, when sequenced in the reverse direction by T7 polymerase terminator (T7TER), K153N was not observed. Plasmid DNA transformation into E.Coli BL21(DE3) cells. Following transformation into BL21(DE3) cells, colonies were observed for both hCAII mutants (V142D and H64A). A random colony was chosen to be cultured and then was induced to express protein with 270 ÃŽÂ ¼M IPTG and 0.1 mM ZnSO4. SDS-PAGE for protein expression. Protein expression was tested with SDS-PAGE. The expected molecular weight of V142D hCAII is approximately 29.2 kDa and the expected molecular weight of H64A hCAII is approximately 29.1 kDa. SDS-PAGE bands are observed between the ladder markers 25.0 kDa and 35.0 kDa for both mutant proteins (Figure , Figure ). Figure : SDS-PAGE loaded with V142D hCAII proteins to examine protein expression. Samples were loaded in different volumes of protein to ensure gel visualization. Lane 15 contains the Fermentas protein molecular ladder and lane 1-4 contain the following: 1 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 4 ÃŽÂ ¼L à ¢Ã¢â€š ¬Ã¢â‚¬â„¢IPTG, 1 ÃŽÂ ¼L+IPTG, 4 ÃŽÂ ¼L +IPTG. All 4 samples had some form of protein expression between 25.0 to 35.0 kDa. Figure : SDS-PAGE loaded with H64A hCAII protein to examine protein expression. One sample was loaded with 4 ÃŽÂ ¼L of H64A protein and +IPTG in lane 10. Lane 6 contains the Fermentas protein molecular ladder. The one H64A sample loaded showed an expression between 25.0 and 35.0 kDa. Calculation of pure protein concentration and extinction coefficient. Following affinity purification and dialysis, pure protein concentration was calculated from UV absorption measurements at 280 nm and the known extinction coefficient of hCAII as 50070 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1 (Table ). The final concentration of the samples of V142D and H64A hCAII were 3.2 ÃŽÂ ¼M and 37.6 ÃŽÂ ¼M respectively. Table : UV absorption measurements at 280 nm of purified protein and the resulting final concentration Mutant Average A280 Protein concentration (ÃŽÂ ¼M) V142D 0.01583 3.2 H64A 0.1884 37.6 SDS-PAGE to assay purity and check approximate molecular weight. Several samples were loaded into the SDS-PAGE for each mutant protein: lysate and wash fractions (collected from affinity chromatography), 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. For H64A, a visible band was only observed for the 10 ÃŽÂ ¼g sample (Figure ). The band was located between the 35 kDa and 25 kDa markers on the ladder. For V142D, none of the 4 samples resulted in a band on the gel (Figure ). Figure : SDS-PAGE shown for H64A mutant protein. Lane 1 contains the Fermentas protein molecular weight marker. Lane 11-14 contains H64A samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. Only the 10 ÃŽÂ ¼g protein had (faint) observable bands located between the 25 and 35 kDa markers. Figure : SDS-PAGE shown for V142D mutant protein. Lane 4 contains the Fermentas protein molecular weight marker. Lane 12-15 contains V142D samples of the following (in order): lysate, wash fraction, 2 ÃŽÂ ¼g protein, and 10 ÃŽÂ ¼g protein. No observable bands are seen for any of the samples. Mass spectrometry. ESI-MS was not successful in analyzing the molecular weight of intact and digested protein of both mutants. A MALDI spectrum was able to be generated for the digested proteins; however, without the digested ESI spectrum to compare to, the peaks from the MALDI spectrum can only be speculatively assigned. Kinetics: Hydrolysis of PNPA. Using the molar absorption coefficient of PNP (1.73ÃÆ'-104 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1cmà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), the rate of each reaction was determined. The predicted rate was calculated using the Michaelis-Menten kinetics: . The plot of predicted rates and actual initial rates vs. PNPA concentration can be seen in Figure , Figure , Figure for wild-type, H64A, and V142D hCAII respectively. The Vmax and KM values for each enzyme were calculated by minimizing the square difference between the predicted and actual reaction rates, and the kcat was calculated using the equation: (Table ). Table : Calculated Michaelis-Menten parameters for wild-type, H64A, and V142D hCAII catalyzing the hydrolysis of PNPA. Wild-type hCAII H64A hCAII V142D hCAII Vmax (ÃŽÂ ¼M/sec) 1.202 0.812 0.218 KM (mM) 1.280 1.957 8.362 kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.141 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢3 2.159 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 6.825 ÃÆ'- 10à ¢Ã¢â€š ¬Ã¢â‚¬â„¢2 kcat/KM (Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 4.02 11.032 8.162 Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for wild-type hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for H64A hCAII enzyme. Figure : Michaelis-Menten plot of initial rate vs. concentration of PNPA added for V142D hCAII enzyme. Kinetics: CO2 hydration. Initial velocity (V0) values were calculated by measuring the progression of the reaction (via concentration of protons) with time (Table , Table , and Table ). kcat values were then calculated using the same equation as in the hydration of PNPA and averaged for the individual enzymes (wildtype, H64A, and V142D hCAII) in a particular buffer (i.e. TRIS or imidazole). Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by wild-type hCAII in TRIS buffer and imidazole buffer. Wild-type concentration (nM) V0 for WT+TRIS (M/s) V0 for WT+Imidazole (M/s) 0 1.3E-08 6.05778E-08 1.5 1.1E-08 N/A 2.5 1.1E-08 5.63E-08 5 2.1E-08 5.16E-08 12.5 5.9E-08 5.63E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 5.3 ±1.62 12.44 ±9.19 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by H64A hCAII in TRIS buffer and imidazole buffer. H64A concentration (nM) V0 for H64A+TRIS (M/s) V0 for H64A+Imidazole (M/s) 12.5 1.4E-08 6.57E-08 25 1.4E-08 5.8E-08 50 1.7E-08 7.53E-08 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.67 ±0.39 3.03 ±1.97 Table : Initial velocity (V0) and kcat values calculated for the hydration of CO2 by V142D hCAII in TRIS buffer. V142D concentration (nM) V0 for V142D+TRIS (M/s) 12.5 6.2E-09 25 5.4E-09 50 5.5E-09 Average kcat (sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) 0.27 ±0.19 Fluorescence detection of ligand binding. DNSA was titrated with H64A hCAII to determine its affinity for the enzyme. The dissociation constant, KD, for DNSA was determined to be 0.086 ÃŽÂ ¼M when protein concentration was 0.25 ÃŽÂ ¼M. Competitive titration of H64A-DNSA hCAII with AZ was attempted, but was not successful as DNSA binding was too tight, making it difficult to be displaced by AZ. Molecular modeling. Literature models of wild-type (PDB code 1CA2) and H64A (PDB code 1MOO) hCAII were analyzed. There is no available structure of V142D hCAII at present. The secondary structure of wild-type is composed of 18 ÃŽÂ ²-sheets (77 residues) and 10 ÃŽÂ ±-helices (42 residues), with the majority of the ÃŽÂ ±-helices falling in the domain of right-handed helices, while very few show left-handed helical properties according to the Ramachadran plot. It also seems that the active site is solely composed of ÃŽÂ ²-sheets, and no ÃŽÂ ±-helices (Figure ). Analyzing PDB structure 3HS4 (AZ bound hCAII), the mechanism as to how AZ inhibits hCAII function can be seen. AZ has 3 binding sites, 2 are novel binding sites and the other provides a mechanism of inhibition. AZ binds the zinc directly at the active site, displacing crucial ligands needed for catalysis. There were some discrepancies found between the crystal structure of H64A [1MOO] as cited on PDB and virtually mutated H64A from wild-type hCAII, resulting in a RMSD (root mean square deviation) of 0.29 Ã… (Figure ). Since no literature structure of V142D is available, no comparison between virtual and crystal structures could be made. Figure : Secondary structure of wild-type hCAII overlain with ribbon to visualize the higher arrangement. Figure : RMSD between H64A hCAII virtually mutated and literature crystal structure. Blues denote the same or similar residues, while reds and oranges indicate completely different amino acids. Discussion Agrose gel results were only visible for samples that contained 20 ng of the plasmid template DNA, rather than the 10 ng plasmid. This may be a result of more amplification during PCR with the 20 ng plasmid, and so would intensely be more visible. Though the 20 ng samples showed bands at the appropriate 6000 bp mark, there was also a faint band that can be seen near the end of the gel. This may be due to non-specific primer annealing. Quantification of DNA purity was done by exploiting the peak absorbances of protein and DNA. DNA maximally absorbs at 260 nm, while protein dominantly absorbs at 280 nm. The purity ratio reports the relative amount of DNA compared to protein present in the sample. The purity of both mutants were approximately 1.8, which is regarded as a relatively pure sample; however, a purity ratio of more than 2.0 would have been ideal. The restriction enzyme digest showed 2 bands (7000, 6000 bp) for all samples, which may have been a sign of poor mixing/ pipetting since the volumes of restriction enzyme were extremely small amounts. If this is the case, only some of the DNA was nicked and some were not, which would result in 2 bands. It was expected that the plasmid sample would have a high band (supercoiled), each of the singly digested samples would have a slightly lower band (nicked), and the doubly digested would show 2 bands that indicated the fragment size of 892 and 5126 bp. Sequencing results showed that a protein mutation occurred when the sample was sequenced in the forward direction by the T7 polymerase. A lysine at position 153 had mutated to glutamine (K153N). However, this mutation was not observed when the T7 polymerase terminator was used to sequence the sample in the reverse direction. A mutation that occurs in one sequencing direction and not the other is usually attributed to sequencing errors, which may be the reason in this case. The SDS-PAGE bands for protein expression coincided with the expected molecular weight for both mutants, which could suggest that the correct proteins were expressed; however, there is a possibility that the proteins expressed could be of similar weight, but completely different. Interestingly, the V142D samples that did not include the protein inducer, IPTG, had a more intense band than the faint ones found for the samples that did include IPTG. This may just be a result of mislabelling. The SDS-PAGE performed to assess purity after the purification process. Mutant V142D had low protein expression as evidenced by its concentration of 3.2 ÃŽÂ ¼M. The V142D mutant should have very low protein expression according to Fierke et al. (1991) because valine at position 142 is uniquely required for maximal expression in E.Coli. It is suggested that by altering position 142, protein stability decreases [2]. Therefore, the protein that was expressed in the previous SDS-PAGE gel may not be V142D hCAII at all. The sample may have been small fragmented contaminant proteins that would have completely run off the gel altogether. However, the low concentration of V142D after purification may also be a major factor in the lack of gel bands observed as well. Unlike V142D, H64A hCAII concentration should not have affected its lack of bands because it was calculated to have had a reasonable concentration of 37.6 ÃŽÂ ¼M. There were some problems loading the samples into the wells; t his could be an explanation as to no observable gel bands. ESI-MS is dependent on concentration because it affects the size of primary droplets [8]. The unsuccessful determination of molecular weight of V142D hCAII may be attributed to its low concentration. The H64 hCAII mutant was also not able to be successfully analyzed with ESI-MS. A possible reason for the failure was that it was not kept on ice while it was not being used. The enzyme may have become inactive and degraded into smaller fragments. This would explain the ESI-MS output obtained for H64A. No definite molecular mass was determined, but the spectrometer did detect a lot of small protein fragments in the sample, all under 1000 amu. The kinetic values obtained from PNPA hydrolysis do not follow similar trends found in literature [2]. The kcat/KM for wild-type hCAII (2500 ±200 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) was found to be significantly larger than V142D hCAII (3 ±0.3 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) in literature, more than 800ÃÆ'- larger [2]. Experimental calculations yielded kcat/KM for V142D (8.16 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1) to be about 2ÃÆ'- larger than wild-type (4.02 Mà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1sà ¢Ã¢â€š ¬Ã¢â‚¬â„¢1), which did not follow literature patterns. The literature trends make more biolog

Role Of Metal Ions In Biochemistr

Role Of Metal Ions In Biochemistr A metal is a chemical element that is a good conductor of both electricity and heat and forms cations and ionic bonds with non-metals. In chemistry, ametal (from Greek ÃŽÂ ¼ÃƒÅ½Ã‚ ­Ãƒ Ã¢â‚¬Å¾ÃƒÅ½Ã‚ ±ÃƒÅ½Ã‚ »ÃƒÅ½Ã‚ »ÃƒÅ½Ã‚ ¿ÃƒÅ½Ã‚ ½ mà ©tallon, mine]) is an element, compound, or alloy characterized by high electrical conductivity. In a metal, atoms readily lose electrons to form positive ions (cations). Those ions are surrounded by delocalized electrons, which are responsible for the conductivity. The solid thus produced is held by electrostatic interactions between the ions and the electron cloud, which are called metallic bonds.[2] Metal ions play essential roles in about one third ofenzymes . These ions can modify electron flow I a substrate or enzyme, thus effectively controlling an enzyme-catalyzed reaction. They can serve to bind and orient substrate with respect to functional groups in the active site, and they can provide a site for redox activity if the metal has several valence states. Without the appropriate metal ion, a biochemical reaction catalyzed by a particular metalloenzyme would proceed very slowly, if at all. The enzyme provides an arrangement of sidechain functional groups having an appropriate sized hole with the preferred groups on enzyme side chains needed to bind the required metal ion. The optimal number of such binding groups is chosen for the particular metal ion, together with the appropriate hydrophobic or hydrophilic environment in the binding site. Metal ions may be bound by main-chain amino and carbonyl groups, but specific binding is achieved by the amino acid side chains, particularly the carboxylate groups of aspartic and glutamic acid, and the ring nitrogen atom of histidine. Other side chains that bind metals ions include tryptophan (ring nitrogen), cysteine (thiol), methionine (thioether), serine, threonine, tyrosine (hydroxyl groups), and asparagine and glutamine (carbonyl groups, less often amino group . No set of general rules exists that describes how a given metal ion will behave in an enzyme . Now that many crystal structures of proteins are being studied by X-ray diffraction, information on the binding of metal ions in the active sites of enzymes is available and should provide clues to the mechanism of action of the enzyme.The examples of catechol methyltransferase andmandelate racemase will be discussed later in this article.The work described here includes results fromexaminations of the crystal structures in the CambridgeStructural Database and the Protein Databank . Astudy of binding, however, also involves an analysis ofthe energetic consequences of changing the way thebinding occurs, so that the most stable binding pattern fora given group of ligands can be deduced. We haveapproached this using ab initio molecular orbital and density functional calculations . In this way weobtain both the binding geometry of ligands and theenergetic consequences of changing this binding m ode. Properties of metal ions Metal ions are generally positively charged and act as electrophiles, seeking the possibility of sharing electron pairs with other atoms so that a bond or charge-charge interaction can be formed. They behave rather like hydrogen ions (the poor mans metal). Metal ions, however, often have positive charges greater than one,and have a larger ionic volume so that they can accommodate many ligands around them at the same time. In addition, metal ion concentrations can be high atneutral pH values, while hydrogen ion concentrations are, by the definition of pH, low at these values. Ligands are the atoms or groups of atoms that are bonded to the metal ion, generally in an electrostatic manner. They are usually neutral or negatively charged and they donate electron density to the metal ion. Thecoordination number of a metal ion, that is, the number of ligand atoms bound to it, is viewed in terms of concentric spheres; the inner sphere containing those atoms in contact with the metal ion, the second sphere containing those in contact with the inner sphere ligand atoms. The number of atoms in these spheres will depend on the size of the metal ion and the sizes of the ligand atoms. For example, sodium is smaller than potassium, and sulfur is larger than oxygen. Measurements of metal ion-liganddistances in crystal structures led to the idea of atomic and ionic radii [9-11]; anion radii can also be derived from the minimum anion-anion distances in crystal structures. The radius ratio, a concept introduced by Goldschmidt [11], is the ratio of the radius of the cation to that of the anion and is generally less than 1.0 Tetrahedral structures have a radius ratio between 0.225 and 0.414, while octahedral structures have a ratio between 0.414 and 0.645. For example, the radius of Mg2+ is 0.65 D, while that of O2- is 1.40 D and their radius ratio is 0.464; the packing is octahedral. The charge distribution in the active site of an enzyme is designed to stabilize the transition state of the catalyzed reaction relative to that of the substrate. In enzyme-catalyzed reactions it is essential that the reactants be brought together with the correct spatial orientation, otherwise the chance of the reaction taking place is diminished and the reaction rate will be too low.The electrostatic environment in the active site is a major factor that serves to guide the substrate to the binding site in the correct orientation. Metal ions can assist in this process, often binding groups in a stereochemically rigid manner, thereby helping to control the action of the enzyme. Thus, an enzyme will bind its substrate in such a manner that immobilization and alignment, ready formation of the transition state of the reaction to be catalyzed,and then easy release of the product will result; metal ions often help in accomplishing this process. Each metal ion has its own chemistry. An example of the differing reactivities of metal cations is provided by their ability to bind or lose water molecules. The exchange of coordinated water with bulk solvent by various cations has been categorized into four groups: those for which the exchange rate is greater than 108 per second including alkali and alkaline earth metal ions(except beryllium and magnesium), together with Cr3+,Cu2+, Cd2+, and Hg2+. Intermediate rate constants (from 104 to 108 per second) are found for Mg2+ and some of the divalent first-row transition metal ions. Those with slow rate constants (from 1 to 104 per second) include Be2+ and certain trivalent first-row transition metal ions. The inert group with rates from 10-6 to 10-2 per second containsCr3+, Co3+, Rh3+, Ir3+, and Pt2+. One of the factors involved in rates of exchange is the charge-to-radius Ratio; if this ratio is high the exchange rate is low.An important reaction catalyzed by metal ions inenzymes is the ionization of water to give a hydrated hydrogen ion and a hydroxyl anion. Initial studies of this process will be discussed here as they are relevant to the action of a metal ion in providing a hydroxyl group and a hydrogen ion for use in an enzymatic reaction. Polarizing Potential of Various Ions Atoms or groups of atoms are considered polarizable if, when they are placed in an electric field, a charge separation occurs and a dipole is acquired. This deformability or polarizability is measured by the ratio of the induced dipole to the applied field. Those atoms that hold on less firmly to their electrons are termed more polarizable. It is found that if two ions have the same inert gas structure (potassium and chloride, for example), the negatively charged anion is more polarizable than the positively charged cation, which holds on to its electrons more tightly. The word hard has been introduced to indicate a low polarizability so that the electron cloud is difficult to deform (like a hard sphere). By contrast soft means high polarizability so that the electron cloud is readily deformed . A hard acid or metal cation holds tightly to its electrons and therefore its electron cloud is not readily distorted; its unshared valence electrons are not easily excited. Soft (polarizable) metal cations contain electrons that are not so tightly held and therefore are easily distorted or removed. A hard acid prefers tocombine with a hard base, while a soft acid prefers to bind with a soft base by partially forming covalent bonds .The type of binding is related to the highest occupied molecular orbital (HOMO) of the electron-pair donor (a lewis base, the ligand) and the lowest unoccupied molecular orbital (LUMO) of the electron-pair acceptor (a Lewis acid, the metal ion). If these have similar energies, then electron transfer will give a covalent (soft) interaction, whereas the energy difference is large, electron transfer does not readily take place and the interaction is mainly electrostatic (hard-hard). Hardcations include the alkali and alkaline earth metal ions while soft metal ions include Cu 2+, Hg2 2+, Hg2+, Pd2+. Inbiological systems, hard ligands generally contain oxygen while soft ligands contain sulfur. Hard acids tend to bind hard bases by ionic forces, while soft acids bind soft bases by partially forming covalent bonds. These hard-soft categorizations are a help in understanding the relative binding preferences of various cations. Most metal ions of biological significance are hard or intermediate between hard and soft. Most soft metal ions and soft ligands are poisonous and they interact with other soft species in the body. For Pb2+ the harder ligands are found in hemidirected structures and the softer ligands in holodirected complexes.Nature has devised many enzyme systems in which a metal ion interacts with the oxygen of a water molecule.If a water molecule can be dissociated into a hydrogen ion and a hydroxyl group, the latter can serve as a nucleophile in chemical a nd biochemical reactions.Nature has chosen activation of a water molecule as a means to obtain such a nucleophile in situation so that a chemical reaction can occur in a stereochemically controlled manner in the active site of the enzyme. The questions we ask are as follows: 1) how does nature ensure that the specific water molecule will be activated; 2) how does nature compensate for the lower water activation power of some cations over others (since a wide variety of metal ions may not be available in the particular active site and the enzyme has to do the best it can with what is available); and 3) how does nature ensure that the required reaction occurs. Ab initio molecular orbital and density functional calculations have been carried out to measure the extent to which a series of metal cations can, on binding with water, cause it to be dissociated into its component hydrogen ions (subsequently hydrated in solution) and hydroxyl ions. Initial data indicate that the charge of the metal ion plays a significant role in modifying the pKa of water. The binding enthalpies of a wide variety of metal ion monohydrates, M[H2O]2+ , have been published [21] but their deprotonation enthalpies are still under investigation. Geometry of Metal-Ion Binding to Functional Groups The geometries of metal ion-carboxylate interactions have been studied in order to determine the following: 1)which lone pair of an oxygen atom in a carboxylate group, syn or anti, is preferred for metal cation binding; 2) does the metal ion lie in the plane of the carboxylgroup; and 3) under what conditions do metal ions share both oxygen atoms of the carboxylate group equally? We found that cations generally lie in the plane of the carboxylate group . The exceptions to this mainly include the alkali metal cations and some alkaline earth cations; these metals ionize readily and form strong bases so it is not surprising that they have less specific binding modes. When the distance of the metal cation to the carboxylate oxygen atoms is on the order of 2.3-2.6 D, the metal ion tends to share both oxygen atoms equally. Otherwise one oxygen atom of the carboxylate group is bound to the metal ion and the other is not. Calcium ions often form bidentate interactions, while it is less common for the smaller magnesium ions. Imidazole groups in histidyl side chains of proteins bind metal ions in a variety of enzymes. One imidazole can, by virtue of its two nitrogen atoms, bind one or two metal ions, depending on its ionization state and the suitabilities of the metal ion. The bases in DNA can also bind metal ions. We have analyzed hydrogen bonding to and from nitrogen atoms in nitrogen-containing heterocycles for crystal structures in the Cambridge Structural Database. It was found that for hydrogen bonding, a slight out-of-plane deviation of the binding atom often occurs. Metal ions bind more rigidly in the plane of the imidazole group. The energetic cost of such deviations were analyzed by ab initio molecular orbital calculations. In an investigation of protein crystal structures in the Protein Databank it was found that the binding of metal ions to histidine in proteins is more rigid and the location of the metal ion is more directional. Thus, if an enzyme needs to control the location and orientation of a carboxylate or imidazole group, it can accomplish this better with a metal ion than by hydrogen bonding. Metal ions in proteins are often involved in structural motifs. When a metalloenzyme carries out its catalytic function it uses one of a few possible three-dimensional arrangements of functional groups around the metal ion to ensure the specificity of the required biochemical reaction. Thus, if such catalytic metal-binding motifs can be identified and categorized, then incipient reactivities of enzymes could be inferred from their three-dimensional structures. Such a categorization, however, requires an understanding of the underlying chemistry of any metal ion in the active site. One motif identified in the crystal structure of cobalt(II) formate consists of a carboxyl group in which one oxygen atom is bound to the metal ion and the other is bound to metal-bound water, to give a cyclic structure. This motif has been found in many metalloenzyme crystal structure , such as D-xylose isomerase . The roles of these motifs are of interest. The metal ion-hydrated-carboxylate motif (I) is planar and commonly found. It does not, however, affect the ability of the metal ion (in studies of Mg2+ complexes) to ionize water. On the other hand, for magnesium ions (which generally have a rigid octahedral arrangement of binding groups) it utilizes 2 of the 6 coordination positions and therefore serves to orient the arrangement of ligands, an effect we have labeled coordination clamping. Motif (II) is also found in several crystal structures such as that of the -subunit of integrin CR3 . It appears to help bind subunits together. A third motif (III) is found in D-xylose isomerase and involves two metal ions with several carboxylate ligands and a histidine ligand . The metal site that binds only oxygen atoms can bind substrate in place of the two water molecules and orient the substrate. The second metal ion site (with histidine as one ligand) then positions a metal ion-bound water molecule to attack the substrate. Roles of Metal Ions in Enzyme Action The crystal structure of mandelate racemase with bound p-iodomandelate provides a useful example of the importance of a metal ion in a reaction . The enzyme binds a magnesium ion by means of three carboxyl groups. The substrate mandelate has displaced water from the magnesium coordination sphere and binds by means of its carboxylate group and an a-hydroxy group.The magnesium ion will lie in the plane of the carboxyl group, as shown by our studies of metal ion-carboxylate interactions . The magnesium holds the substrate firmly in place so that the catalytic abstraction and addition of a hydrogen atom by His 297 or Lys 166 is precisely effected . The magnesium probably also aids this activity by affecting the electronic flow in the carboxylate and hydroxyl groups by mild polarization. We have found that metal ion coordination is better than a hydrogen bond in aligning a functional group; there is considerable flexibility in a hydrogen bond as we found for imidazoles . In the reaction c atalyzed by the enzyme mandelate racemase the magnesium ion binds substrate . A Histidine (His 297) and Lysine (Lys 168) are positioned to abstract a hydrogen ion from the substrate and, if it is added again from the other side, racemization occurs. Hydrogen bonding to a carboxylate group of the substrate helps to stabilize an enolate intermediate in the reaction. In catechol O-methyltransferase , a methyl group is transferred from the sulfur of Sadenosy[ methionine to catechol. The magnesium ion is oriented by a motif of type I and it binds substrate in such an orientation that a hydroxyl group is near the S-CH3 group, and the other hydroxyl group is held in place by a carboxylate group. There are many other examples of two-metal ion active sites, such as hemerythrin, alkaline phosphatase and superoxide dismutases (which have been well documented). These studies of the geometries and energetics of metal-ion ligand b inding can therefore aid in our understanding of metalloenzyme function Metals in the RNA worid By combining our limited knowledge of metal-ion-binding to contemporary RNAs and our more extensive knowledge of metal-ion-binding to proteins, it is possible to speculate on the role of metal ions in prebiotic molecular evolution. It seems clear that specifically bound metal ions coevolved with RNA molecules. Many of the mononuclear sites in Table 5 are formed with, or can be engineered into, small RNA fragments. Since such sites are highly hydrated and contain limited direct contact with the RNA, the observed affinities are only moderate, in the 1-1000 ÃŽÂ ¼M range. These sites are also expected to show limited specificity, predominantly dictated by the chemical nature of the ligands. Furthermore, in these examples, the RNA structures themselves are likely to be quite flexible and can accommodate a variety of metal ions with only minor distortions to the overall RNA fold. These minimalist sites are sufficient to stabilize the secondary and tertiary structures observed in these motifs. The metal ion sites generated on small RNAs appear to be capable of facilitating a variety of different types of chemistry. Activities range from the transesterification and hydrolytic reactions of small ribozymes (Pyle 1996; Sigurdsson et al. 1998) to the more exotic porphyrin metalation (Conn et al. 1996) and Diels-Alder condensation reactions (Tarasow et al. 1997) catalyzed by aptamers produced from in vitro selection experiments.These small RNAs have only limited amounts of structure and therefore are likely to position the catalytic metal ions by only a few points of contact. The relatively modest rate enhancements supported by catalytic RNAs such as these probably reflect the types of species that first evolved from random polymerization events. Very active metal ions might have assisted in this process but would have increased the danger of side reactions that would accidentally damage the catalyst. A striking difference between most RNA metal-binding sites studied thus far and those seen in proteins is the degree of hydration. Both structural and catalytic metal-ion-binding sites in proteins are predominantly dehydrated (Lippard and Berg 1995). Water molecules occasionally appear in the coordination spheres of these metal ions, but in these cases, they are often believed either to be displaced by the substrate when it enters the active site or to take part in the catalytic mechanism of the enzyme. Such protein sites also bind their metal ions much more tightly than the RNA systems. In fact, tight binding is a requirement for dehydrated sites, since there is a characteristic energy (ÄHhyd) associated with the hydration of any ion. The net binding energy upon coordination of the ion must account for the energetic cost of dehydration. The question arises, Why are such dehydrated sites not observed in RNAs? One possibility is that metal-binding sites in RNAs are intrinsically different from those in proteins. RNA has a much more limited set of ligands to use in generating a specific metal-binding pocket. Amino acid side chains containing thiols and thioethers are well suited to binding a variety of softer metals. In addition, the carboxylate side chains provide anionic ligands with great versatility in their potential modes of coordination. They can act as either terminal or bridging ligands and bind in either monodentate or bidentate geometries. The nucleotides, on the other hand, are much larger and more rigid than the corresponding amino acids. The anionic ligand in RNA, the nonbridging phosphate oxygen, is an integral component of the backbone and therefore is more limited in its conformational freedom than the aspartate and glutamate carboxylate groups. The heterocyclic ring nitrogens and the keto oxygens from the bases are held in rigidly planar orientations by the aromatic rings. This geometric constraint severely limits the ability of an RNA to compact encompass a metal ion and provide more than facial coordination and therefore complete dehydration. It also explains why the most specific metal-binding sites are not in the Watson-Crick base-paired regions of the structure where the conformation is too constrained. Instead, metalion- binding sites are clustered in regions of extensive distortion from the A-form RNA helices. There is also the question of the folding of RNAs relative to that of proteins. It is possible that in RNAs there is insufficient energy in the folding and metal-binding process to completely displace the waters of hydration around a metal ion. It has been suggested that in contemporary RNAs, modified nucleotides might be present to assist in metal ion binding (Agris 1996). A more straightforward possibility, however, is that most RNAs studied to date are structurally too simple. In these RNAs, most residues involved in metal ion binding are solvent-exposed. Thus, the RNAs have no real inside comparable to the hydrophobic core of a protein. The largest RNA crystallographically characterized to date is the P4-P6 domain. On the basis of that structure, it was proposed that an ionic core may substitute in RNA folding for the hydrophobic core of proteins such that the 3 ° structure assembles around a fixed number of discrete metal-binding sites (Cate et al. 1997). Even in this structur e, however, the most buried of the metal-binding sites are significantly hydrated. It could be that all metal-ion-binding sites in RNA are at least partially hydrated. One can imagine several advantages to using hydrated ions within the ionic core of a large RNA. Hydrated ions would span larger voids than dehydrated ions and allow looser packing of secondary structure elements. The hydrated ion also can accommodate a wide range of structural interactions through its orientation of the water molecules as compared to direct coordination of metal ions at every site. In addition, the energy associated with deforming the outer-sphere interactions should be significantly less than what would be observed for distorting the innersphere coordination. A consequence of RNAs having a core of hydrated ions is that one might expect this core to be much more dynamic than the hydrophobic core of a protein. In the modern protein world, metal cofactors are associated with a variety of reaction types, including electron transfer, redox chemistry, and hydrolysis reactions. Trans esterification and hydrolytic activities, however, are the primary catalytic behaviors observed in ribozymes. Did these other catalytic activities not develop until the dawn of the protein world, or are there undiscovered natural catalytic RNAs that are the ancestors of the early redox enzymes? Through the use of in vitro selection experiments, the scope of RNA catalysis has been significantly broadened is almost certainly capable of catalyzing these other classes of reactions, but it is still unclear whether there are naturally occurring examples. Such an enzyme would likely use a metal ion cofactor other than Mg(II), so the search for RNA molecules that naturally use alternative ions is of significant interest. A recent selection experiment showed that a single base change results in an altered metal ion specific ity for RNase P (Frank and Pace 1997). It is clear from this result that catalytic RNAs retain the ability to adapt to an everchanging environment, using the resources available to evolve and to overcome evolutionary pressures. Were RNAs to have evolved out of an environment devoid of metal ions, they probably would have found a way around the problems of folding and generating reactive functional groups. The primordial soup and all cellular environments that have evolved subsequently contained a variety of ions, however. Given the availability of metal ions, they will certainly play a significant role in the biology of current and future RNAs. Effect of metal ions on the kinetics of tyrosine oxidation by Tyrosinase The conversion of tyrosine into dopa [3-(3,4-dihydroxyphenyl)alanine] is the rate limiting step in the biosynthesis of melanins catalysed by tyrosinase. This hydroxylation reaction is characterized by a lag period, the extent of which depends on various parameters, notably the presence of a suitable hydrogen donor such as dopa or tetrahydropterin. We have now found that catalytic amounts of Fe2+ ions have the same effect as dopa in stimulating the tyrosine hydroxylase activity of the enzyme. Kinetic experiments showed that the shortening of the induction time depends on the concentration of the added metal and the nature of the buffer system used and is not suppressed by superoxide dismutase, catalase, formate or mannitol. Notably, Fe3+ ions showed only a small delaying effect on tyrosinase activity. Among the other metals which were tested, Zn2+, Co2+, Cd2+ and Ni2+ had no detectable influence, whereas Cu2+ and Mn2+ exhibited a marked inhibitory effect on the kinetics of tyrosine ox idation. These findings are discussed in the light of the commonly accepted mechanism of action of tyrosinase. Tyrosinase (monophenol,dihydroxyphenylalanine oxygen oxidoreductase; is a copper-containing enzyme responsible for melanogenesis in plants and animals, which catalyses both hydroxylation of tyrosine to dopa and its subsequent oxidation to dopaquinone (Hearing et al., 1980; Lerch, 1981). The first reaction, which represents the rate-limiting step in melanin biosynthesis (Lerner et al., 1949), is characterized by a lag period that has subsequently been explained in terms of a hysteretic process of the enzyme (Garcia Carmona et al., 1980). The extent of this induction time depends on various parameters including, besides pH and both substrate and enzyme concentration, the presence of a suitable hydrogen donor. Kinetic studies carried out on tyrosinases from various sources (Pomerantz, 1966; Pomerantz Murthy, 1974; Hearing Ekel, 1976; Prota et al Abbreviations used: dopa, 3-(3,4-dihydroxyphenyl)-alanine; SOD, superoxide dismutase. To whom correspondence and reprint requests should be addressed. 1981) have shown that dopa, in very low concentration, is the most effective reducing agent in eliminating the lag period, whereas other catechols, such as dopamine, adrenaline and noradrenaline, behave similarly to ascorbate and NADH and NADPH in only shortening it, even at high concentration. Tetrahydropterin, a well-known specific cofactor of other aromatic hydroxylases (Lerner et al., 1977; Marota Shiman, 1984), is also effective in stimulating tyrosinase activity, although to a lesser extent than dopa. At present, no other organic or inorganic substances have been reported to shorten or lengthen the lag period of tyrosine oxidation. Although metal ions are known to play a role in many biologi cal processes, little attention has been directed to their possible involvement in melanogenesis, particularly in the early enzymic stages .As a part of our continuing studies on the chemistry of melanin pigmentation (Prota, 1980; Sealey et al., 1982; Palumbo et al., 1983), we report the results of a survey on the effect of metal ions on the activity of purified Sepia tyrosinase, readily available in large amounts from the ink of the cephalopod Sepia officinalis thermostability of amalyse Three Metal Ions Participate in the Reaction Catalyzed by T5 Flap Endonuclease*à ¢- ¡ Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a two-metal-ion mechanism. Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of>4 AËÅ ¡ appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2_ or Mn2_). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions. Key cellular processes such as DNA replication, DNA repair, RNA processing, and RNA degradation require the rapid hydrolysis of the phosphate diester linkages of nucleic acids. The uncatalyzed hydrolysis of phosphate diesters under biological conditions is an extremely slow process with an estimated half-life of 30 million years at 25  °C (1). Protein nucleases and RNA enzymes produce rate enhancements of 1015-1017 to allow this reaction to proceed on a biologically useful time scale. Most enzymes catalyzing phosphate diester bond hydrolysis have a requirement for divalent metal ions. Based largely upon crystallographic observations, most metallonucleases are proposed to catalyze reactions using a two-metal-ion mechanism (Fig. 1a) analogous to that suggested for the phosphate monoesterase alkaline phosphatase (2, 3), although this view is not universally accepted. Three recent reviews present contrasting views on the roles of metal ions in protein nuclease and RNA enzyme reactions and illustrate this controversy (4-6). One family of metallonucleases over which there has been considerable mechanistic debate are the flap endonucleases (FENs)3 (7-12), which are present in all domains of life and play a key role in DNA replication and repair. Unlike most metallonucleases, which typically possess a cluster of three or four active site carboxylates, the FEN active site is constructed from seven or eight acidic residues located in similar positions in FENs from a range of organisms (Fig. 1b, see also supplemental Fig. S1) (7, 9, 10, 13-16). Several FEN x-ray structures also contain two active site carboxylate-liganded divalent metal ions, designated as metals 1 and 2 (9, 13-15). The position of metal 1 is similar in all cases, but the metal 2 location varies. In all but on