Biochemistry 461, Section I Your Printed Name:___________________________________May 6, 1997Exam #3 Your SS#:___________________________________Prof. Jason D. KahnYour Signature:___________________________________You have 80 minutes for this exam.Exams written in pencil or erasable ink will not be re-graded under any circumstances.Some information which may be useful is provided on the bottom half of this page.Explanations should be concise and answer the specific question asked. You have more space on theexam than is necessary—don’t think you need to fill the page.You will not need a calculator for this exam. No other study aids or materials are permitted.Possibly Useful Information:Michaelis-Menten equation: v0 = Vmax[S]/(Km + [S])Type of inhibitionApparent KmApparent VmaxApparent Vmax/KmCompetitiveaKmVmax(1/a) Vmax/KmUncompetitive(1/a¢)Km(1/a¢)VmaxVmax/KmMixed(a/a¢)Km(1/a¢)Vmax(1/a) Vmax/KmNoncompetitive (a=a¢)Km(1/a) Vmax(1/a) Vmax/Kma = 1 + [I]/KI a¢ = 1 + [I]/KI¢21. (20 pts) Fundamentals of Enzymatic Catalysis.(a; 8 pts) List four types of enzymatic catalysis and give very brief descriptions for two of these.(b; 4 pts) Why are enzyme active sites typically in pockets or clefts on the enzyme surface?3(c; 8 pts) Citrate was proposed in the 1930’s to be a critical intermediate in the Krebs cycle, which iscentral to metabolism. Citrate is achiral, as two substituents (the CH2COO- groups) of the centralcarbon atom are the same. It is converted to isocitrate as shown, by the enzyme aconitase. Laterinvestigators (1940’s) were surprised to find that an isotopic label on citrate (with the label indicatedby the star) ended up in only one of the two possible places in isocitrate. Why did they initially,incorrectly, expect that label should go to both of the indicated positions? Propose a simpleexplanation for the observed specificity and draw a picture to illustrate your answer. The answer hasto do only with the binding of citrate to the enzyme surface, not with the mechanism of aconitase.Hint: if the reaction were just acid or base-catalyzed, both products would be observed.CaCOOHOCH2COOCbH2COO*CaCOOHCH2COOCb*OHH COOCitrateIsocitrate (Fisher projection)CaCOOHCH2COOCb*OHH COONOT OBSERVEDOBSERVEDAconitaseThe “Ca” and “Cb” carbons are the same ones in each structure.42. (25 points) Enzyme Kinetics.(a; 7 pts) Given the Michaelis-Menten equation on the front page, derive the double-reciprocal(Lineweaver-Burk) expressions used to linearize v 0 vs. [S] data, and sketch the resulting plot. Labelthe axes, the slope, and the x and y intercepts.(b; 5 pts) What is the operational definition of Km (i.e. how is it measured)? What is Km in terms ofthe three elementary rate constants of the Michaelis-Menten equation? Why is it that measuring aKm is useful even when the actual mechanism is more complicated than the M-M mechanism?5(c; 7 pts) In the box below, fill in the species and equilibria needed to extend the Michaelis-Mentendescription to include mixed inhibition. Indicate which equilibrium is relevant to pure uncompetitiveinhibition, and explain why it is rare to find a pure uncompetitive inhibitor for a simple M-M enzyme.E+S ES E+P(d; 6 pts) Why is it not catalytically advantageous to have an extremely stable ES (or EP) complex?What point along the reaction coordinate should be bound most tightly to make an efficient enzyme?Sketch a free energy reaction coordinate diagram to illustrate.63. (20 points) Enzymatic Reaction Mechanisms.(a; 10 pts) Di-isopropylfluorophosphate (DIFP) is a potent irreversible inhibitor of serine proteases.The phosphorus atom is extremely electrophilic due to the presence of the electronegative fluorine,which is also a good leaving group. Complete the mechanism below for the reaction between Ser 195and DIFP. Explain why DIFP does not react with other serines on the protein. (You can answer thelast part even if you were unable to draw the mechanism).Ser195OHPOOFODIFPIntermediateProductHF7(b; 6 pts) Serine proteases still catalyze peptide hydrolysis (slowly) when Ser is replaced by Ala!1. What principle of enzymatic catalysis explains this observation?2. Explain why replacement of the other two residues of the catalytic triad with alanine does notfurther reduce the reaction rate.3. What class of artificial enzymes work according to the same principle?(c; 4 pts) What do cofactors do in general? Name a cofactor and describe what kind of reaction it isused for.84. (15 points) Regulation of Enzyme Activity.(a; 9 pts) By analogy to cases mentioned in lecture, guess what the main kind of regulation used tomodulate each of the following biological processes is, and state why:1. Changes in the activity of threonine deaminase in response to leucine levels, where leucine is thedownstream product of the reaction catalyzed by the enzyme.2. The response of signal transduction intermediates to the hormone epinephrine, which stimulatesglycogen breakdown.3. The development of the Drosophila body plan, whereby an embryo is elaborated into a fly.(b; 4 pts) Why is ATCase activated by ATP and inhibited by CTP? What kind of inhibition is this?9(c; 2 pts) Why are serine proteases synthesized as inactive zymogens, which must be proteolyzed tobecome active?5. (20 points) Carbohydrates and Lipids.(a; 8 pts) Name three functions for carbohydrates in biochemistry, and the characteristics ofcarbohydrates which make them especially suited for each of these functions.(b; 5 pts) When single-tail detergent concentrations exceed the critical micelle concentration, addeddetergent makes more micelles, not bigger micelles. In contrast, double-tail phospholipid formsbilayers which tend to get larger and larger. Explain these observations.10(d; 7 pts) Briefly describe the experiments which show that transverse diffusion of phospholipids inmembranes is slow and that lateral diffusion is fast.Do Not Write Below This LineScore: Question 1: out of 20Question 2: out of 20Question 3: out of 25Question 4: out of 15Question 5: out of 20Total: out of