Answer Key1CHEM/BIOC/MMG 205October 23, 2006BIOCHEMISTRY IEXAM IIRead the questions carefully.Extra pages are available. Put your name on every page.This exam is a closed-book, closed-notes exam. No outside material may be usedand you may not discuss the exam with anyone else.Due to conflicts with the exam schedule, not all students are taking the exam at thesame time. Do not discuss the exams until they are handed back.The exam has 6 questions for a total of 60 points. The exam duration is 2 hours.Good Luck!Answer Key21. (12 points) What classes of enzymes perform the following transformations(a) oxidation-reduction reactions oxidoreductases(b) transfer of functional groups transferases(c) hydrolysis reactions hydrolases(d) addition to double bonds lyases(e) isomerization reactions isomerases(f) formation of bonds with ATP cleavage ligasesAnswer Key32. (9 points) Double reciprocal plots of bisubstrate enzyme mechanisms are shownbelow. Indicate which mechanism gives rise to which plot.Ordered single displacement orrandom single displacementwhere A influences B binding.Random single displacement.A has no effect on binding Band vice versa.Double displacement(ping-pong)1/[A]1/v01/v01/v01/[A]1/[A][B][B][B]2[B]2[B]2[B]3[B]3[B]3[B]Answer Key43. (10 points) Mutation of asp102 in the catalytic triad of trypsin to asn (D102N)leads to a 100-fold reduction in the Vmax of the enzyme, although the mutantenzyme continues to display burst-phase kinetics.(a) Explain the observed reduction in VmaxTrypsin operates by a ping-pong mechanism in which the second step is rate-determining. A reduction in Vmax, means the second step has been slowed. Thesecond step is hydrolysis of the covalent enzyme-product acyl intermediate.Asp102 orients his57 by H-bonding to one of the imidazole ring nitrogens. Theother nitrogen of his57 is a general base that deprotonates water as it attacks theacyl intermediate.The D102N mutation does not allow the formation of the H-bond to his57, so therate determining step is slowed, and the measured Vmax is smaller.(b) Analysis of the X-ray crystal structure of the D102N mutant indicates that thespecificity pocket, oxyanion hole and orientation of the nucleophilic ser195 areidentical to the wild type protease. On the same axes, sketch the double-reciprocal plots you would expect for the wild-type enzyme and the D102Nmutant.All the factors that influence substrate binding and product dissociation areunchanged, so Km remains the same. Vmax is reduced 100-fold.1/v01/[S]-1/Km10.01wild-typeD102NAnswer Key54. (8 points) The initial rates of an enzyme-catalyzed reaction change with pHaccording to the following profile. Given that the mechanism involves ageneral acid and a general base, what two amino acids might perform thesefunctions? Briefly explain your reasoning.Activity is low at pH less than 4, and increases at pH greater than 4. This suggeststhat a carboxylate group COO- is necessary for activity (no activity: COOH belowpKa). COO- is a general base, so the partner must be a general acid (RH+). Theactivity falls off above pH 6, so we’re looking for an amino acid with a side chainpKa of about 6. Histidine would be a good choice. The general base could beaspartate or glutamate.2 3 4 5 6 7 8v0pHAnswer Key65. (11 points) The model for Michaelis-Menten kinetics is as follows:E + S EX‡ E + PA simple model of positive homotropic allostery is that substrate binding at asite remote from the active site increases substrate binding at the active site,increasing k1 according to:k1app = c[S]k1where c is a constantShow that this model leads to non-hyperbolic Michaelis-Menten kinetics.Assuming steady state in [EZ‡] and measurements of initia rates at [P] = 0, wecan write the Michaelis-Menten equation as:v0 = Vmax[S]/([S] + (k--1 + k2)/k1)Substituting for k1appv0 = Vmax[S]/([S] + (k--1 + k2)/c[S]k1)Rearrangingv0 = Vmax[S]2/(K + [S]2)where K = (k--1 + k2)/ck1A plot of v0 vs [S] is a sigmoid, not a rectangular hyperbola.k1k-1k2Answer Key76. (10 points) What are the advantages and disadvantages of covalent regulation ofenzyme activity versus allosteric regulation?Allosteric RegulationAdvantages: Allosteric effectors are metabolites that reflect the prevailing cellularconditions. Effector binding is reversible, and adjusts enzyme activity to themomentary needs of the cell. Allosteric enzymes are rapidly controllableaccording to effector concentration.Disadvantages: Allosteric control is determined by the amount of effector at anygiven moment, so the enzyme can’t stay “on” if the effector concentration ismomentarily reduced.Covalent RegulationAdvantages: Covalent regulation (zymogens, phosphorylation/dephosphorylation)is rapid and reversible in the case of regulation by phosphorylation. Theprocess is efficient (ie many enzymes can be regulated simultaneously) becausethe regulating enzymes (kinases and phosphatases) are catalytic. Enzymefunction can be decoupled from cellular conditions.Disadvantages: The effector enzymes are metabolically costly, and often form partof elaborate enzyme cascades (blood clotting factors are a good example).Covalent regulation may be irreversible, as in the case of