Unit 4 1 UNIT 4 PART A: ENZYME MECHANISMS PART B: PROTEIN EVOLUTION PART C: CONTROL OF ENZYMATIC ACTIVITY NOTE: The unit 4 and 5 tests are combined! Please be sure to prepare both units before you try to tackle the quiz! PART A: ENZYME MECHANISMS Asignment: Nelson & Cox, review pp. 188 - 192, pp. 192 - 194, 204 - 213. In Unit 3 we learned that enzymes form an ES complex when appropriate substrates make weak noncovalent interactions with amino acid side chains. This gives the complex its specificity. Once the ES complex has formed, weak noncovalent interactions are optimized to stabilize the transition state and alow the reaction to proced. In this section we wil learn how specific catalytic groups contribute to catalysis using thre enzymes (chymotrypsin, hexokinase, and enolase) to ilustrate the points. Unit 4 2 Objectives: 1. Binding energy contributes to reaction specificity and catalysis. a. Discuss the following forces that are involved in the binding of substrates to enzymes. For each, point out whether the interacting groups must be precisely aligned to get significant binding and whether the distance betwen interacting groups is critical for binding. 1) electrostatic interactions 2) hydrogen bonds (Fig. 2-5, p. 46) 3) van der Wals forces 4) hydrophobic interactions b. Discuss the importance of binding energy, ?G B , in enzyme catalyzed reactions (pp. 188 - 192). c. Discuss each of the following in terms of binding energy (p. 192). 1) entropy reduction 2) desolvation 3) induced fit 2. Specific catalytic groups contribute to catalysis (pp. 192 - 194) a. Describe general acid-base catalysis. What is the general purpose of acid/base catalysis (pp. 192 - 193)? Use Fig. 6-9 (p. 193) to identify the amino acid side chains that can function as acid-base catalysts. b. Describe covalent catalysis. What is the purpose of covalent catalysis (p. 193)? c. Many enzymes require metal ions for activity. Describe two functions of these metal ions (pp. 193 - 194). Unit 4 3 3. Enzyme activity is afected by pH (p. 204) a. List two reasons why enzyme activity is afected by pH (p. 204). b. In a folded protein, nearby amino acid side chains can influence the pK of a specific side chain. Discuss the efect of a positive microenvironment on the pK of aspartic acid. Wil this positive microenvironment have the same afect on the pK of histidine? Discuss the efect of a negative microenvironment on each of these side chain types. (Note: The book does not give the answer to the above objective. The general idea, though, is that the presence of a positive charge decreases the local proton concentration and so other groups in the vicinity respond to a pH that is higher than the "bulk" value). 4. The catalytic mechanism of Chymotrypsin (pp. 205 - 209, especialy Fig. 6-21, pp. 208 - 209) a. Describe the 2° and 3° structure of chymotrypsin (Fig. 6-18, p. 206). b. Name the thre residues in the "catalytic triad" and discuss their position in the 1° and 3° structure (Fig. 6-18, p. 206). c. Using Fig. 6-21 (pp. 208 - 209) as a guide, discuss the mechanism of action of chymotrypsin. (Note you should be able to name the important amino acids, but you do not need to memorize their position in the 1° sequence.) 1) What is the purpose of the "hydrophobic pocket"? 2) What is the role of each of the residues in the "catalytic triad" in the overal mechanism? 3) Draw the structure of the intermediate. What interactions stabilize this species? What is the name of the region where these interactions occur? 5. What is a transition state analog? Discus the types of evidence that suggest enzyme - transition state complementarity (Box 6-3, pp. 210 - 211). Unit 4 4 6. Use Fig. 6-22 (p. 212) to discuss the role of "induced fit" in the mechanism of Hexokinase. 7. Use Fig. 6-23 (p. 213) to demonstrate the roles of Mg + , Lys 345, and Glu 211 in the mechanism of Enolase. Note: You wil learn about the biological roles of hexokinase and enolase in Unit 10 of this course! PART B: PROTEIN EVOLUTION Genomes today can be quite large and complex, yet they evolved from simpler genomes that contained fewer genes and thus encoded fewer proteins. Where did these "new" genes come from? Many are the result of divergent evolution. In this proces, ancestral genes were duplicated. One copy of the gene continued to encode the same protein, the other was fre to mutate. Sometimes the duplicate gene was mutated such that it became inactive, becoming a pseudogene. Some of these mutations occur in the regions that do not encode protein but instead occur in regions which determine when and in which tisues this protein is made. There are excelent examples of this in the globin gene family in which diferent globin genes are expresed during diferent stages of development. Other mutations lead to subtle changes in protein structure and/or function. Asignment: Nelson & Cox: pp. 29 - 36, 102 - 107. 1. Use Fig. 1-32 (p. 30) to describe the evolution of protein families (also refer to Study Guide section introduction). 2. Use Fig. 1-34 (p. 31) to discuss the "RNA world" scenario. 3. Define the following terms: a. genome (p. 34) b. homolog (pp. 34, 104) Unit 4 5 c. paralog (pp. 34, 104) d. ortholog (pp. 34, 104) e. alignment (pp. 104 - 105) f. pseudogene (Study Guide section introduction) 4. Use Fig. 3-31 (p. 105) to define "signature sequence" and to describe how it can be used to determine how closely two taxonomic groups are related to one another. 5. Breifly describe how homologous sequence information can be used to generate a phylogenetic tre like the one shown in Fig. 3-32 (p. 106). a. What is represented by the "internal nodes"? b. What is the significance of the length of the line that connects two nodes? PART C: CONTROL OF ENZYMATIC ACTIVITY In cel metabolism, groups of enzymes work together in pathways to cary out sets of chemical reactions. In each system there is at least one enzyme that limits the rate of the overal proces because it catalyzes the slowest (or rate-limiting) reaction. These "regulatory enzymes" increase or decrease their catalytic activity in response to signals within the cels. By the action of such regulatory enzymes, the rate of each metabolic sequence is constantly adjusted to provide the metabolites required by the cel. Metabolic pathways are often regulated at the commited step in a pathway. Asignment: Nelson & Cox, pp. 220 - 228. Unit 4 6 1. Briefly, discuss the thre major clases of regulatory enzymes in metabolic pathways clearly distinguishing betwen each clas (p. 220). 2. Alosteric Enzymes (pp. 220 - 222) a. Compare alosteric enzymes to others in terms of structure (p. 220). b. Nelson & Cox use aspartate transcarbamoylase (ATCase) as a model of an alosteric enzyme. It consists of both catalytic and regulatory subunits and catalyzes the commited step in the biosynthesis of pyrimidine rings for DNA and RNA. How does ATCase difer from hemoglobin with respect to the types of subunits (Fig. 6-32, p. 221)? c. Discuss the kinetic properties of alosteric enzymes (p. 222). 1) In a cel, what is the key advantage of any enzyme with sigmoid versus hyperbolic kinetics (p. 222)? 2) Discuss the thre substrate-activity curves shown in Fig. 6-34 (p. 222). 3) Review the meaning of homotropic and heterotropic alosteric control (p. 222). 3. Reversible Covalent Modification (pp. 223 - 225) a. Using Fig. 6-35 (p. 223) as a guide, list several types of covalent modification. b. Phosphoryl groups afect the structure and catalytic activity of many proteins (pp. 224 - 225) 1) Name the clas of enzymes that ataches phosphoryl groups to specific amino acids (p. 224). 2) Name the clas of enzymes that removes phosphoryl groups (p. 224). Unit 4 7 3) Name the thre amino acid side chains on which both of the above enzyme types act (p. 224). What functional group do these thre side chains have in common? 4) Explain why the addition of a phosphoryl group results in altered enzyme activity (p. 224). 5) Discuss the regulation of glycogen phosphorylase by covalent modification (pp. 224 - 225). 4. Regulation by proteolytic cleavage (pp. 226 - 227) a. Distinguish betwen zymogens and proenzymes (pp. 226 - 227)? b. Use Fig. 6-38 (p. 227) to explain how chymotrypsinogen is activated? c. Since this type of activation is ireversible, how are proteolyticaly activated enzymes inactivated (p. 227)? Jim Blankenship Microsoft Word - U04_F08.doc
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