METABOLIC BIOCHEMISTRY Immo E Scheffler Winter 2002 MIDTERM EXAM ANSWERS All answers are to be written into the Blue Book Leave the first inside page blank for scoring There are 11 questions Make sure that each answer is clearly identified with the question number at the top or left side of the page Useful Information Avogadro s number 6 02 x 1023 molecules mole 1 Faraday 96 494 Coulomb mole 96 494 Joules Volt mole Gas constant R 8 31 Joules K 1 mol 1 1 987 cal K 1 mol 1 0 082 liter atm K 1 mol 1 1 calorie 4 184 Joules QUESTION 1 12 min The following table from your textbook lists a variety of coenzymes some of which have not been covered in class A series of structures are given below Identify the structure by its name from the Table 8 2 and give an example of a reaction in which the coenzyme is involved The reaction requires only the names of substrates and products not their structural formulae A the cofactor shown in thiamine pyrophosphate it is required in decarboxylation reactions for example in the formation of ethanol and CO2 from pyruvate B the cofactor is nicotinamide adenine dinucleotide NAD used in a variety of oxidation reduction reactions for example the oxidation of glyceraldehyde 3 P to 1 3 bisphosphoglycerate with inorganic phosphate from the medium The lactate dehydrogenase or pyruvate dehydrogenase reactions are also acceptable C the cofactor is pyridoxal phoshate so far we have seen it appear in the glycogen phosphorylase reaction QUESTION 2 8 min a For an enzyme obeying Michaelis Menten kinetics a plot of initial rate vs substrate concentration yields a characteristic curve Give the algebraic expression describing this curve in terms of S KM vmax and explain how such data allow one to estimate KM quite readily By measuring the rates at various substrate concentrations up to very high values one can obtain Vmax from the asymptote A horizontal line at 1 2 Vmax intersects the experimental curve at Km i e when S is equal to Km we observe half of the maximal velocity b In the same diagram contrast the behavior described in a with a curve one would obtain for an enzyme exhibiting allosteric behavior no algebra or formulae required The curves become distinctly S shaped and different curves are obtained with different concentrations of effector QUESTION 3 12 min There are four boxes below labeled A D a match them in pairs i e describe which kinetic curves correspond to which type of inhibition b characterize name the types of inhibition shown A represents competitive inhibition and it matches the curves shown in D B represents uncompetitive inhibition and it matches the curves shown in C c give a short one or two sentences explanation for the observation that all the lines intersect in one point on the Y axis in figure D In competitive inhibition the inhibitor and the substrate bind to the same site Thus at infinite substrate concentrations x 0 in the double reciprocal plot all rates are the same regardless of the inhibitor concentration a single intercept with the y axis In uncompetitive inhibition substrate and inhibitor bind to different sites and even at infinite substrate concentrations the inhibitor can still lower the rate i e the intercept on the y axis moves up as more and more inhibitor is present QUESTION 4 12 min a 5 points Give the structural formula for NAD show also the reduced form with only the business end of the molecule b 4 points Illustrate the use of this cofactor in the final reactions of fermentation production of alcohol structural formulae required for the substrates intermediates and products but not the co factors The answer should show the decarboxylation of pyruvate to acetaldehyde and CO2 and the reduction of acetaldehyde to ethanol with NADH as cofactor Structural formulae are required c 3 points If 1 14C pyruvate is given to a yeast extract would you expect to be able to obtain radioactive ethanol Explain your answer with the help of the structural formulae in the answer for b In the structural formula for pyruvate the carbons should be numbered the carboxyl group is the 1 carbon The carboxyl group becomes the carbon dioxide hence no radioactive ethanol can be produced The following tables give the standard free energies of hydrolysis for a variety of phosphorylated compounds and the standard reduction potentials for biologically important half reactions They are to be used for Problems 5 and 6 QUESTION 5 12 min Consider the following three reactions and determine which if any of these reactions could take place spontaneously as written from left to right with reagents present at standard conditions The reaction that could occur as determined from thermodynamics nevertheless does not occur in cells What could be a reason a ATP creatine ADP phosphocreatine b Phosphoenolpyruvate glucose pyruvate glucose 6 phosphate c AMP PPi ATP a reaction would not be spontaneous under standard conditions since the free energy of hydrolysis of phosphocreatine in higher than the energy of hydrolysis of ATP phosphocreatine has the higher high energy phosphate b thermodynamically this reaction is highly favored Go 0 however there is no enzyme in the cell that we have discussed or that I know that can catalyze such a reaction c this reaction is highly unfavorable Go 10 9 kcal mole as written QUESTION 6 8 min The following are some reactions involving the oxidation and reduction of carbon compounds Which of the following would proceed in the direction shown under standard conditions provided the appropriate enzymes were available a Malate NAD oxaloacetate NADH H b Malate pyruvate oxaloacetate lactate a the half reactions are i oxaloacetate 2H 2e malate E o 0 166 volts ii NAD H 2e NADH E o 0 320 volts if the first reaction is reversed and added to the second we obtain the overall reaction in a E o would still be negative and hence this reaction would not proceed under standard conditions however if the NADH is quickly removed and its concentration is kept low we can see this reaction as shown for the Krebs cycle b the half reactions are i oxaloacetate 2H 2e malate E o 0 166 volts ii pyruvate 2H 2e lactate E o 0 185 volt again reversing the first reaction and adding it to the second yields the overall reaction E o would still be slightly negative and hence the reaction would not go under standard conditions but it could be pushed either way by slight changes in the concentrations of the reactants and products Both answers require a calculation of E o for
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