Alcohol Dehydrogenase: From Ethanol To Acetaldehyde

Alcohol Dehydrogenase: From Ethanol To Acetaldehyde (20) 1. Alcohol dehydrogenase (AD) is an enzyme which catalyzes the reaction of its natural substrate ethanol to form acetaldehyde. The Km of AD, from rhinoceros livers, for ethanol is 1 X 10-3M. This enzyme is however somewhat non-specific and will recognize substrates other than ethanol. How would the kinetic plot be affected if AD were to separately catalyze methanol and isopropanol instead of ethanol? Assume that the overall Vmax remains the same in all 3 cases. How would the Km change for methanol compared to ethanol (higher, lower, the same)? How would the Km change for isopropanol compared to ethanol (higher, lower, the same)? How would the Kms of methanol and isopropanol compare (which higher than the other or about the same). Based upon your knowledge of the mechanisms by which enzymes work, briefly explain how you decided to place your new Kms. Hint: The alcohols are being added separately. There is not any kind of competition between the alcohols. They are not included in the same reaction. For your reference, the structures of these alcohols are below. Because ethanol is the natural substrate of Alcohol dehydrogenase (AD), AD would have a higher affinity and bind more readily to ethanol than other alcohols, including methanol and isopropanol. Because AD has a higher affinity for ethanol than other alcohols, its Km would be lower than methanol and isopropanol. The lower the Michaelis constant (Km) the less substrate required to get to  ½ Vmax or  ½ the maximum reaction rate and the higher the affinity of the enzyme for the substrate. Higher Km means more substrate concentration to reach  ½ Vmax and less affinity of the enzyme for the substrate. Vmax or the maximum reaction rate can be approached, but never actually reached. The Km for methanol would be higher than ethanol, thus requiring more substrate to reach  ½ Vmax and demonstrating lower affinity of AD for methanol. The Km for isopropanol would be higher than ethanol, thus requiring more substrate to reach  ½ Vmax and demonstrating lower affinity of AD for isopropanol . The Km for methanol would be lower than the Km for isopropanol and show a higher affinity for AD. The Michaielis-Menten kinetic plot would reflect a Km of 110-3M at  ½ Vmax for ethanol, a Km greater than 110-3M for methanol and a Km greater than the Km of methanol for isopropanol. The overall Vmax is the same for all three, so the  ½ Vmax for all three will stay the same. The plotted curve would become less vertical with the initial angle for ethanol becoming more acute and the curve becoming more linear as it changed from ethanol to methanol to isopropanol. Ethanol is ADs natural substrate, so based on enzyme mechanisms, it is able to bind more readily to AD due to its size and shape which fits ADs active site and allows ethanol to get close enough to create hydrogen bonds. The substrate and enzyme change confirmation and become destabilized which stabilizes the transition state, lowers the energy of activation and allows easier formation of the reaction products. Methanol and isopropanol do not bind as well, likely due to their structure or size and shape. Methanol is one carbon shorter which would prevent it from fitting in the AD site as well as ethanol and has fewer numbers of hydrogens, reducing H-bonding potential. Isopropanol is one carbon larger than ethanol which might make it too bulky to effectively bind to AD. Isopropanol is a secondary alcohol, with two carbon atoms attached to the carbon bonded to the OH, creating a bulky Y shape and not a chain alcohol like methanol and ethanol. This conformation and bulky shape prevents isopropanol from binding more readily than methanol, which is similar to ethanols linear shape. (10) 2. Briefly explain the protein cleavage involved in the maturation of an insulin molecule from proinsulin. Briefly explain 3 reasons why it is important that insulin be made as an inactive precursor requiring editing. Hint: Think in terms of things that would be important to the action of insulin (decreasing blood sugar). Protein cleavage is post-translational processing. Proinsulin is the precursor to insulin. Proinsulin is a polypeptide chain that loops around to form two disulfide bonds between four cysteine amino acids, two near either end. Endopeptidase cuts two molecules by proteolysis to remove the middle portion of the polypeptide. The final disulfide stabilized protein is insulin. Inactive proinsulin allows for optimal intracellular insulin stores that can be edited or activated quickly if needed to lower blood sugar and quickly prevent hyperglycemia. Proinsulins can be produced rapidly in response to elevated blood sugar with the post-translational processing switched off quickly; leaving the inactive molecules, once blood sugar is under control. Proinsulin is important because it is not degraded until it is needed, thus does not cause harmful low blood sugar levels and maintains sustained basal levels of insulin in the body. (10) 3. Briefly and individually outline the mechanisms of action for covalent, competitive, non-competitive, and uncompetitive enzyme inhibitors indicating how they effect enzyme action. For each type of inhibitor, describe a unique example of how we could learn something valuable, and at least somewhat practical, about an enzyme from each type of inhibitor study. The mechanism of action for covalent enzyme inhibitors is covalent binding in the enzyme active site and thus preventing substrate binding. This is irreversible and completely deactivates the enzyme requiring more enzymes to be produced to catalyze the reaction. This could tell us what amino acids bind in the enzyme active site by identifying covalent inhibitor modified functional groups and also substrate binding order. The mechanism of action for competitive enzyme inhibitors is they are shaped like the substrate and can bind in the enzyme active site, blocking the substrates binding. Competitive inhibitors can be outcompeted by increasing the substrate concentration and are reversible. Competitive inhibitors could be used to determine enzyme substrate affinities by finding out how much substrate is required and how long it takes to get back to  ½ Vmax. The mechanism of action for non-competitive enzyme inhibitors is they bind in a place other than the enzyme active site, allowing the substrate to bind, but they destabilize the transition state which hinders the enzyme by obstructing its proper performance and reducing Vmax. Non- competitive inhibitors are reversible, but cannot be outcompeted because they do not bind to the active site. Non-competitive inhibitors could be used to determine an enzymes induced fit mode of action as the substrate would still be able to bind, but not fully react. The mechanism of action for uncompetitive enzyme inhibitors is the substrate and inhibitor bind together in multi-substrate enzymes. While substrate binding and Km seem better, velocity is less because the inhibitor acts as part of the substrate. They are reversible. Uncompetitive inhibitors could be used to determine effective drug therapies by inhibiting an enzyme to varying degrees without permanently altering it, counter acting large amounts of the multi-substrate enzyme but not eliminating it from performing other useful functions. (10) 4. In discussing advances in molecular biotechnology, we mentioned 2 processes whose names sound remarkably similar called RFLP and AFLP. These two processes indeed share some similarities, but have many differences. Briefly explain 2 significant similarities that these share in their processes. Briefly explain 2 significant differences in terms of what these processes are used for. One similarity in RFLP and AFLP processes is cutting DNA for RFLP and cDNA for AFLP with restriction enzymes to create fragments. Another similarity is that DNA is electrophoresed in RFLP to separate different sized restriction fragments creating unique patterns for organisms or individuals (with the exception of twins) much like fingerprints and used for comparison. PCR products are electrophoresed in AFLP to compare tissues, experiments or expression profiling. One difference in what these processes are used for is RFLP is used to compare DNA from people or organisms for genetic fingerprinting and forensics, and AFLP is used to profile gene expressions (requiring mRNA to be converted to cDNA) of uncharacterized tissues, organisms or experiments. Another difference is AFLP can be used for Quantitative Trait Loci which help identify multifactorial inheritance of traits and assist in genome mapping, whereas RFLP is not used for QTL, but can be used for identifying a persons predisposition for a particular disease. (10) 5. Life on the planet Zornock encodes its genetic info in overlapping nucleotide triplets such that the translation apparatus shifts only one nucleotide at a time. In other words, if we had the nucleotide sequence ABCDEF on Earth this would be two codons (ABC DEF) whereas on Zornock it would be 4 codons (ABC, BCD, CDE, DEF) and the beginning of two others. Briefly explain and compare the effect of each of the following types of mutations on the amino acid sequence of a protein in 1) an earthling and 2) a Zornocker. A. The addition of one nucleotide. B. The deletion of one nucleotide. C. The deletion of 3 consecutive nucleotides. Assume these all occur in the middle of a gene. X = added nucleotide, ? = unknown nucleotide A1. One nucleotide added resulting in ABCXDEF in the earthling would create a frameshift that would produce the original codon ABC, a new codon XDE and one codon beginning F. A2. One nucleotide added resulting in ABCXDEF in the Zornocker would create one new codon, making a total of 5 codons, (ABC, BCX, CXD, XDE, DEF) and the beginning of two other codons EF? and F. B1. The deletion of one nucleotide resulting in ABCEF in the earthling would create a frameshift that would produce one original codon, ABC and two different beginnings EF? and F. B2. The deletion one nucleotide resulting in ABCEF in the Zornocker would result in 3 complete codons, ABC, BCE and CEF and two beginnings EF? and F. C1. The deletion of three consecutive nucleotides resulting in ABF in the earthling would create a frameshift that would result in one new codon, ABF. C2. The deletion of three consecutive nucleotides resulting in ABF in the Zornocker would result in one new codon and two partial codons, ABF and the beginnings BF? and F. The insertions and deletions in the earthling would produce a frameshift, creating different codons and a different polypeptide chain from the mutation on. Other effects of the frameshift could be inserting a different AA into the polypeptide or stopping translation altogether. These genotype effects could create non-functioning proteins or fragments, partially functioning proteins or no protein expression. The insertions and deletions in the Zornocker would add or remove codons at the site of the mutation, but would not alter the polypeptide chain after the mutation due to the overlapping nucleotide triplets. (10) 6. Imagine that weve isolated a new and potentially useful mutation in an existing model plant. Our goal as biotechnologists might be to characterize the mutation, figure out what protein it affects, figure out how it is expressed, figure out how it is controlled, and how to best take advantage of it for crop improvement. Using the techniques that weve covered so far, briefly outline a series of experiments and expected results, using at least 5 of the techniques weve discussed, to attempt to achieve the above goals. Hint: There is more than one way to do this. 1 In order to characterize the mutation, we could use Sanger DNA sequencing to determine the amino acid sequence of the mutated gene. We use a primer and DNA polymerase to start DNA synthesis. We then prepare reactions with dideoxynucleotides (ddNTP) for each nitrogenous base, A, T, C and G. We run the reactions with normal nitrogenous bases and one ddNTP nitrogenous base representing either A, T, C or G. The ddNTPs terminate the DNA chains and when all the reactions are electrophoresed on a gel with lanes A, T, C and G, we can read from the bottom up to determine the DNA sequence. We could then compare the DNA sequence to the sequence of the existing model plant to determine the differences in amino acid sequences caused by the mutation. 2. In order to characterize what protein it affects, we could detect gene expression and protein interactions by using qRT-PCR. First we create mRNA by transcribing the mutant DNA genes. Next, we convert the mRNA using reverse transcriptase to cDNA. Then we run a qPCR on the cDNA and add SYBR green to the products. SYBR green intercalates the DNA and we can measure the fluorescence and determine the number of mRNA copies, thus determining which proteins are affected. 3. In order to figure out how it is expressed, we could use DNA microarray and protein microarray analysis. With DNA microarrays we obtain gene chips and hybridize fluorescently labeled cDNA from the tissues containing the mutation. The mutation sample is compared to the model sample in parallel microarrays. A machine then analyzes and overlays the images to measure transcript levels, identify products and determine upregulation and downregulation of many proteins. We could also use protein microarrays which are similar to DNA microarrays, but are used to identify other proteins and compounds a protein interacts with. At times, protein function can be inferred by analyzing the environment in which it is expressed. 4. To figure out how it is controlled, we could use in situ hybridization to locate the mutant gene expression products or RNA molecules produced. First we chemically fix sample tissues to slides. With DNA probes we could localize mRNAs to see which cells and where in these cells the gene is being expressed. We could probe with antibodies to determine which proteins are being translated. We could add or subtract associated enzymes, substrates and cofactors and alter internal and external cell conditions to see how this changes the gene expression and thus determine how the gene is controlled. 5. To determine how best to take advantage of it, we could genetically engineer the model plant with the mutation by inserting the mutant DNA into a Ti plasmid, creating a recombinant Ti plasmid, and have Agrobacterium introduce that into the model plant. The Ti plasmid would recombine with the model plant DNA and create a genetically engineered plant that expresses the new trait. We could then run various experiments on the genetically engineered plant to determine if the trait is expressed as desired and if not, change the variables until we get the advantage we are looking for.

Festival: Meaning of Life and Festivals Essay

A festival is an event which is celebrated collectively for celebrating a cause. Festivals are mostly celebrated to propagate the cultural heritage of nations. Festivals highlight various aspects of a society. For example people in all around the world celebrate Christmas, New Year, Sports events such as Olymics, Easter, Eid and other religious and cultural events to exhibit their emotions and happiness. Festivals at national level help to promote solidarity and patriotic spirit in the society. Religious festivals all around the world bring convergence and propagate acceptance of all kinds of religions in different geographical segments. International festivals help to increase brotherhood and remove ethnic racism in the world. Owing to the fact that world has become a global village, celebrations of all kinds of festivals in a country improves the overall image of the country and shows the acceptance of cultural diversity in the country. They help keeping alive the older traditions and provide entertainment to the people celebrating events in different parts of the world.All the festivals that we celebrate have some profound meaning in it and they have some pragmatic purpose also. If we take the example of Christmas, we all know that why it is being celebrated but when it comes to the purposes that it serves along with the religious one; primarily it is a psychological one. We all need some days of break in our life from our regular job. Sometimes we take it voluntarily but mass don’t understand the purpose of taking break and it is for these people, the festivals serve better that anybody. They are forced to enjoy life and thus they can improve the quality of their life. There are some other purposes also that a festival serves and usually you will find that people meet each other along with their families and thus improve their social life. So now you can understand the meaning of celebrating a festival and we expect that you will also spread the message celebration the world over.

Blood Brothers Essay

My initial example is where Eddie is advising Mickey what to say when he asks Linda out. Eddie: Linda my loins are burning for you. Let me lay my weary head on your warm breast; Linda, I love you, I want you, the very centre of my being calls out for you! Eddie’s environment has obviously affected the way that he could just think of something to say which is very romantic. You could not imagine Mickey saying such things. Eddie is more confident and knows how to use his vocabulary well in order to get people to do things. When Mickey tries to, his outcome is abysmal. Mickey: Erm†¦ er, well the thing is the centre of my†¦ being cries out for†¦ erm, my knees, loins are†¦ (snaps) Linda for Christ’s sake will you go out with me? Linda agrees. Mickey cannot find the right words to say even if he is told what to say. Mickey’s vocabulary is retarded because of his environment. His school is worse than Eddie’s so he tries to copy Eddie but still cannot do that. I put this under humour because I thought it was quite funny but it could also go under language as both of the boys language has been affected by their environment. My next example is when the policewoman asks Eddie what he is doing. Eddie replies â€Å"Adolf Hitler†. Then the policewoman asks him what his name is and he says that he is waiting for the 92 bus. For one moment I think Eddie wanted to be like Mickey and tried to be cheeky to the police officer but because of his family and environment he was unable to do that and it ended up the wrong way around. This example is like my original but the other way around. Now, I am going to discuss the way that Russell uses plot to stress the point that family and environment have an effect on peoples lives and opportunities. The whole of the story is based around two males who are separated at birth. One of the twins had been told that there was a twin brother and that he died at birth, the other had been told that he was an only child. Now they are isolated from each other their lives begin to change because of their background and environment. My first example is when Mickey and Linda are married and are looking for a house from the council. They had been waiting for approximately five years for a new house and still had not got one! When Linda was passing the Council Offices she stepped into the housing officer’s office and asked to speak to him. When he arrived Linda was surprised as the man was Eddie. She pleaded once again for a house to live in, and because of Eddies environment he said that all he needed to do was to make one phone call. Then Mickey and Linda would be at the top of the list and could have a house very soon. Because of Mickey and Linda’s environment, they were forced to go to the council for a cheap or free house and considering Eddie’s status he could do this extremely quickly. My second example for plot is where Linda turns out just like Mrs Johnson, (Mickey’s mum. ) In â€Å"Educating Rita,† another of Russell’s work, Rita was expected to have a baby as soon as they got married. This is just like Linda with Mickey. In their family and environment they are expected to have a baby young and to traditionally stay around the house. This reduces their opportunities in life as they do not work once they have had a baby. The man of the house, in their environment, is foreseen to do all of the work and bring all of the money in. This is exactly what happens in this story. Now, I have completed â€Å"plot†, I will go on to analyse the staging devises Willy Russell uses to show the effect that family and environment have on the two boys lives. There was a recent performance at a local theatre, which I went to see. I found that by going to see the acted story I found how even the way you dress, your family and your environment were involved in some way. My leading example through â€Å"set† is costume. I noticed Mickey was wearing clothes too big for him which have most likely been passed down through the family. Eddie is the complete opposite to Mickey in the way that he is wearing smart, new school clothes and looks very sharp in his uniform. Russell shows how even clothes have an effect on family and environment. At the play it showed the brothers in completely different houses. One at the rough end of the village and the other at the very high end of social environment. They live in different ways and houses because of their environment. Their family either reduces the opportunity to live in a high class house or improves the opportunity. There are certain changes in the acted story, the first being where Mickey helped his brother complete a crime which went wrong. People in Mickey’s environment often resort to this as they need the money, but people in Eddie’s environment would never be constrained to doing this sort of thing as they always have money. When Mickey went to jail he had to be put on anti-depressants to help him cope with the stress and loneliness of prison. This was the final straw for Mickey because all of his life he had been pushed to the limit craving for food and money, lots of things also go wrong in Mickey’s environment. When he finally reached confinement he felted that was enough. If Eddie had been placed in jail he would probably be able to cope as he has had less to put up with than Mickey but would most likely be bailed anyway! I conclude that every little detail which Willy Russell has put into his well crafted book, and performance, has something to do with family and environment. Because of his own experiences he was able to complete the task of writing this book and include very graphic details of how family and environment effects peoples everyday lives and opportunities.

Gun Control Is Unconstitutional - 1069 Words

Gun Control Is Unconstitutional Is it right to guarantee 100% of your rights outlined in Constitution of the United States of America in one area, but only about 90% in another area? The Second Amendment allows citizens the right to bear arms.This topic is very important to every traditional and patriotic American that wants to provide and protect for their families. Americans need to see that guns are not the trouble for all the violence and crimes that are reported on the news. Guns are needed for protection and owning a gun is not unconstitutional. Some people will argue that the US Constitution allows citizens to bear arms only for a well regulated militia, A militia being an army composed of ordinary citizens. This is true that†¦show more content†¦National Crime Victimization Survey data,†robbery and assault victims who used gun to resist were less likely to be attacked†. John Lott, PhD,and David Mustard. PhD, which claim â€Å"more guns equal less crime†. This shows that you don t even need to harm someone to protect yourself . We need guns for safety of our families at home or on the street and to protect our personal property we have worked hard for. From my own experience, my neighbor pulled a loaded gun on a intruder and didnt even need to shoot for the intruder to obey. Nobody was harmed and nobody was killed; the intruder just left. Moreover, there are dangerous animals at least in California that I know of roaming wild even on residential streets. For example my Dad’s friend had a Mountain lion attack his pigs and he had to grab his shotgun to protect his livestock and when he went outside the Mountain lion charged at him and he had to shoot the Dangerous Mountain lion. Events similar to this are very common for Americans living in country and rural areas, that need to protect themselves and their livestock. Self defense is most respected and better for the community then being a victim. It is up to you for the safety of yourself and your loved ones. In addition, many Traditional Americans have a cultureShow MoreRelatedGun Control Is Constitutional Or Unconstitutional892 Words   |  4 Pagesdifferent viewpoints when it comes to if gun control is Constitutional or Unconstitutional, Blek tends to focus on why there should be restrictions and gun laws in place while Sobran emphasis that the right to bear arms should not be invaded or limited. Both men highlight some specific topics that are actually similar in nature yet, each with their own twist, allowing a person to fully grasp both sides and depict both the positive and negative affect if gun laws where in fact implemented. On theRead MoreAnalysis Of The Article Gun Control Is Unconstitutional `` Essay1723 Words   |  7 Pagesif a strong gun control law is not enforced into the entire nation. For years now President Obama has been demanding stronger gun control but the conservative Republicans have been fighting the bill claiming that it jeopardizes the rights given by the second amendment. In the article â€Å"Gun Control is Unconstitutional†, the author quotes â€Å"in another issue of the Federalist Papers (No. 45), the powers of the federal government were to be â€Å"few and defined†... federal power to regulate guns is anythingRead MoreWhy Gun Control Is Bad793 Words   |  4 PagesGun Control Argumentative Essay All guns have the potential to be dangerous and should be handled with care. Guns have been part of American life for as long as people can remember. For most people guns are perceived as very dangerous and harmful. However, that is not true; guns can be dangerous, but only if they are in the wrong hands. Owning a gun is right that every American should be able to take pride in having. Guns are used for self-defense, hunting, law enforcement and other uses.Read MoreEssay on Gun Control in Canada1400 Words   |  6 Pages  Ã‚  Ã‚   There has been considerable debate recently in Canada over the issue of gun control. The Canadian parliament enacted the Firearms Act to enforce gun control by requiring gun owners to register their firearms. Just recently, the government of Alberta lead in a charge, including five other provinces and numerous pro-gun groups, complaining that the law is unconstitutional and intrudes on provincial jurisdiction. They also claim that the act infringes on property and civil rights that are guaranteedRead MoreThe War on Gun Control Essay957 Words   |  4 Pages Gun Control War We are no stranger to gun control debate and controversy here in the U.S. We hear of gun deaths, mass and school shootings on the news on a daily basis. Is it safe to say that the U.S. needs permanent gun control laws to protect its citizens or would it be in violation the constitution? The second amendment was written by our founding fathers back in 1791 in a day where it was common for any citizen to own a weapon. Many times we tend to misinterpret our rights as well as weRead MoreThe Bill Of Rights Of The United States1557 Words   |  7 Pagesban of handguns was a discrepancy in which the citizens of the city were not able to rightfully exercise their Second Amendment right to bear arms. After the law was looked at by the Supreme Court in DC vs. Heller, the court ruled the law was unconstitutional and citizens living in the District of Columbia were being unjustly denied their constitutional rights. After hearing the Supreme Court’s decision in DC vs. Heller, a 76 year old Chicago resident named Otis McDonald looked to remove a City ofRead More1st and 2nd Amendment1723 Words   |  7 Pagesholds and are tested through legal cases. A great example is found in an important case which tested the freedom of religion clause of the Constitution was the case of Vitale vs. Engel in 1962. In this case the Supreme Court decided that it was unconstitutional to have an educator guide prayer in school, because it conflicted with the Establishment Clause in the First amendments. (â€Å"Engel v† 1962.). Another case which tested the establishment case is the case of Van Orden v. Perry is another exampleRead MoreGun Control Paper1134 Words   |  5 PagesGuns Do Not Kill People; People Kill People Gun control is a very large problem going on in the United States today. All guns have the potential to be dangerous and should be used with precautions, as well as the right safety regulations. Guns can be hazardous and tragic accidents can happen, like the Sandy Hook shooting in Connecticut, but only if they are in the hands of the wrong person. Guns are not only used every day for protection, such as law enforcement like the military and the policeRead MoreGun Control Laws Will NOT Reduce Crime Essay1336 Words   |  6 PagesGun control laws aim to restrict or regulate firearms by selecting who can sell, buy and possess certain guns. Criminals do not obey laws and stricter gun control laws or banning guns will have little effect on reducing crimes. There are many myths about gun control reducing acts of gun violence, which are simply not true according to research. People are responsible for the crimes, not the guns themselves. Taking guns away from United States citizens that u se them for many reasons, shooting practiceRead MoreGun Control859 Words   |  4 PagesScholarly Essay: Gun Control There has been considerable debate recently in Canada over the issue of gun control. The Canadian parliament enacted the Firearms Act to enforce gun control by requiring gun owners to register their firearms. Just recently, the government of Alberta lead in a charge, including five other provinces and numerous pro-gun groups, complaining that the law is unconst... Gun Control Gun control Gun Control Part I:Introduction The issue of gun control and violence