Biochemistry 461 Section I May 6 1997 Exam 3 Prof Jason D Kahn Your Printed Name Your SS Your 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 the exam 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 inhibition Apparent Km Apparent Vmax Apparent Vmax Km Competitive aK m Vmax 1 a Vmax Km Uncompetitive 1 a Km 1 a Vmax Vmax Km Mixed a a Km 1 a Vmax 1 a Vmax Km Noncompetitive a a Km 1 a Vmax 1 a Vmax Km a 1 I KI a 1 I KI 2 1 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 is central to metabolism Citrate is achiral as two substituents the CH2COO groups of the central carbon atom are the same It is converted to isocitrate as shown by the enzyme aconitase Later investigators 1940 s were surprised to find that an isotopic label on citrate with the label indicated by 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 simple explanation for the observed specificity and draw a picture to illustrate your answer The answer has to 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 CH2 COO CH2 COO HO Ca H Ca COO H Cb COO OH COO CH2 COO OBSERVED Isocitrate Fisher projection Cb H2COO Citrate Aconitase H H The Ca and Cb carbons are the same ones in each structure Ca COO Cb COO OH NOT OBSERVED 4 2 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 v0 vs S data and sketch the resulting plot Label the 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 of the three elementary rate constants of the Michaelis Menten equation Why is it that measuring a Km 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 Menten description to include mixed inhibition Indicate which equilibrium is relevant to pure uncompetitive inhibition 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 6 3 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 195 and DIFP Explain why DIFP does not react with other serines on the protein You can answer the last part even if you were unable to draw the mechanism Ser195 O F P O H O O DIFP Intermediate HF Product 7 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 not further 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 is used for 8 4 15 points Regulation of Enzyme Activity a 9 pts By analogy to cases mentioned in lecture guess what the main kind of regulation used to modulate 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 the downstream product of the reaction catalyzed by the enzyme 2 The response of signal transduction intermediates to the hormone epinephrine which stimulates glycogen 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 to become active 5 20 points Carbohydrates and Lipids a 8 pts Name three functions for carbohydrates in biochemistry and the characteristics of carbohydrates which make them especially suited for each of these functions b 5 pts When single tail detergent concentrations exceed the critical micelle concentration added detergent makes more micelles not bigger micelles In contrast double tail phospholipid forms bilayers 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 in membranes is slow and that lateral diffusion is fast Do Not Write Below This Line Score Question 1 out of 20 Question 2 out of 20 Question 3 out of 25 Question 4 out of 15 Question 5 out of 20 Total out of 100