kcat, but isozyme M (in muscle) has a Km of 5 um and isozyme L (in liver) has a Km of 20 um. Answer the following questions about this situation.

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1 ENZYMES and KINETICS (10 points this page) A substrate S is converted into product P. When an enzyme is added to the reaction, the activation energy is lowered. Use single sentence answers for the following: 1) What is the effect of addition of enzyme on the forward reaction (S to P) and the reverse reaction (P to S)? The rate of each reaction is increased to the same extent. That is because the activation energy of each reaction is lowered to the same extent 2) What is the effect of adding the enzyme on the equilibrium constant for S conversion into P? The equilibrium constant for an enzyme catalyzed reaction is precisely the same as that for the uncatalyzed reaction. 3) Suppose that the enzyme lowers the activation energy by 5kJ per mole. What letter best describes the change in rate of the S to P reaction compared to when no enzyme is present? your choice: A (all you really needed to know was that its an expoenential relationship and that lowering the activation energy hastens the reaction ) A) it is faster by a factor of e (5,000/RT) B) it is faster by a factor of (5,000/RT) C) it is slower by a factor of e (5,000/RT) D) the change depends on the original Eact Two versions (isozymes) of the same enzyme convert substrate S into product P. They each have the same kcat, but isozyme M (in muscle) has a Km of 5 um and isozyme L (in liver) has a Km of 20 um. Answer the following questions about this situation. 4) At what substrate concentration will M be half-saturated? M is half saturated when S equals the value of Km 5 um 5) What fraction (between 0 and 1) of saturation will enzyme M have at 20 um substrate? S/(S + Km) = 20/25. So 0.8, or 4/5, or even 80% are acceptable 6) What fraction of saturation will enzyme L have at 20 um substrate? L is half saturated when S equals the value of Km 20 um 7) What is the ratio of maximal rates, Lmax/Mmax, for the two enzymes? (Think first ) The ratio is 1. Since the kcats are the same, the maximal rates (for a given amount of enzyme) are the same. page 1

2 8) (3 points) Write the general reaction for phosphorylation of an enzyme E. Use OH to represent the R group that actually receives the phosphate. Make sure to include all necessary substrates and products, and include the general name of the type of enzyme that catalyzes this reaction kinase E-OH + ATP E-OPO ADP ENZYMES and KINETICS cont (10 points this page) The graph is a picture of a Lineweaver-Burk plot,, with data from two different enzymes, I and II, plotted. 9) (2 points) In this sort of plot, each axis is a function of either substrate concentration or rate. Indicate what each axis represent in the space below The X (horzontal) axis is : 1/[S] The Y (vertical) axis is: 1/[V o ] we also accept 1/[V] II I 10) Which of the two enzymes, I and/or II, follows Michaelis-Menten kinetics? _I_ But I spelled Menten wrong, and in deference to Dr. Menten s memory, here is a web site extolling her under-appreciated accomplishments (and spelling her name right) 11) (2 points) Why do you say the answer in 10)? (One sentence please) The Lineweaver-Burk plot converts the Michaelis-Menten equation into a linear function of the two variables, 1/S and 1/V o 12) Where on this plot does one find the saturated rate for an enzyme? Write below please. rate A E I Allosteric enzymes are often encountered in the study of metabolic regulation. Below is a simple rate plot, in rate plotted against substrate concentration S. Sketch in three curves, making sure to indicate which is which with labels. 13) Draw a curve for a typical allosteric enzyme, label it E [substrate] 14) Draw a new curve for E when an allosteric activator is added, and label it A 15) Draw a third curve for E when an allosteric inhibitor is added and label it I 16) What protein structural feature is nearly always associated with kinetic behavior of this sort? This enzyme almost certainly has multiple subunits and hence, quaternary structure page 2

3 OXIDATION REDUCTION (10 points this page) 17 (4 points) Glyoxylate can be oxidized to the two carbon, dicarboxylic acid oxalate. This is one source of the material in kidney stones. Starting with the structure of glyoxylate below, write a reaction for the oxidation of glyoxyate by NAD. Include any water or H + you need to balance the reaction O H C CO 2 H CO - 2 CO2-2 O NADH 2H + NAD + (Hey! These are the reactions from last year s midterm! ) The following four half-reactions have the standard reduction potentials listed. A) αkg + CO 2 + 2H + +2e isocitrate E o = B) ΝΟ 3 + 2Η+ + 2e NO H 2 0 E o = C) crotonyl-coa + 2Η + 2e butryl-coa E o = D) Pyruvate + 2H+ 2e lactate E o = There are 4 acceptors and 4 donors in this collection of reactions. Answer the following questions 18) Which molecule in this collection of 8 (donors and acceptors) is the best oxidizing agent? NO 3 - is most prone to get reduced, so it is the best oxidizing agent 19) Which molecule in this collection of 8 is the best reducing agent? isocitrate is the most prone to give up its electrons (get oxidized), so it is the best reducing agent 20) List the letters for the reaction or reactions that would spontaneously reduce H + ion at standard conditions Those reactions that would spontaneously run backwards (as written) when connected to an H2/H+ cell are the negative values A, C, D 21) (3 points) For reaction D, show the relationship between E o and the equilibrium concentrations for this half reaction. Use letters for any constants (eg A for avogadro's number) E o = E o = -(RT/nF) ln [pyruvate]/[lactate]) page 3

4 An inborn error of glycolysis (10 points this page) One of the most powerful tools in the study of metabolism is use of radioactive atoms. The next questions will use a labeled molecule to understand glycolysis in the cells of normal individuals, and those with an inherited disease. 22) (2 points) 1-[ 14 C]-fructose-6P is added to a cellular lysate with a fully functional glycolytic pathway. Draw a molecule of labeled pyruvate that would result from the labeled molecule ending up in that product. Just the pyruvate please H 3 14 C 23) (2 points) Does every molecule of pyruvate derived from a labeled fructose have a labeled carbon? Yes or no, and explain your answer No, half of the pyruvate molecules produced come from the unlabled 4,5, 6 carbons of the fructose that are originally produced as glyceraldehyde 3 P C O CO 2-14 CH 2 C CH CH CH CH 2 O OH OH OH 14 CH 2 C CH 2 O OH HC O CH OH CH 2 There is a severe inherited disease caused by a null allele of the triose phosphate isomerase (TPI) gene. You know, the 5 th enzyme of the glycolytic pathway. Homozygotes produce NO TPI enzyme activity. 24) (4 points)write the reaction that TPI catalyzes, including structures of reactant(s) and product(s) 14 CH 2 C CH 2 O OH HC O C OH C 14 H 2 I included the labeled one carbon, for ease of identification. You didn t need to do this 25) (2 points) 1-[ 14 C]-fructose-6P is added to a cell lysate completely deficient in TPI, and glycolysis is allowed to proceed. Is there still label found in pyruvate? Why or why not? No label is found in the pyruvate, because the resulting labeled molecule, 14 C-DHAP can not be converted into G3P, so this lableled product can not continue down the glycolytic pathway. (See picture above) page 4

5 An inborn error of glycolysis (cont ) (10 points this page) 26) (3 points) 1-[ 14 C]-fructose-6P is added to a cell lysate deficient in TPI, what would you predict the main molecule with labeled 14C to be? Justify your answers with a picture and/or a sentence. 14 CH 2 C CH CH CH CH 2 O OH OH OH 14 CH 2 C CH 2 O OH The label will ALL be found in DHAP, which is shown above. The inability of the lysates to then convert the DHAP into G3P will freeze the label at this step. HC O CH OH CH 2 When a molecule of glucose undergoes glycolysis in one of these cells that is completely TPI deficient, the bookkeeping or ATP profit changes in understandable ways. Let s figure this out (so that you don t have to memorize it ). 27) How many ATP are consumed for each glucose that goes down this mutant pathway? 2 28) How many ATP are produced from each glucose that goes down this mutant pathway 2 29) How many reduced carrier molecules are made per glucose by the TPI-deficient pathway? 1 30) What is this reduced carrier molecule called NADH 31) How many molecules of pyruvate are formed per glucose by the TPI-deficient pathway 1 32) (2 points) Explain the number you gave in 31) below with one sentence: Only one pyruvate can be formed, that from the G3P formed in the original cleavage of Fr1,6 bisphosphate. The other three carbon skeleton can not enter the pathway. So one pyruvate. page 5

6 Glucose coming and going (10 points this page) Nature s Sugar Bowl Glycogen is a key glucose storage molecule in mammals. 33) (4 points) Using the picture below of a single non-reducing end of glycogen, write the glycogen phosphyorylase reaction, showing any substrates and products, but don t worry about drawing the remaining glycogen molecule. etc. + PO 4-3 PO ) (2 points) How is the molecule produced by glycogen phosphorylase then put into the glycolytic pathway? Write the relevant reaction, no structure required. (Hint: a mutase is required) Glu-1P phosphogluco mutase Glu-6P 35) (2 points) Glucose regulates glycogen phosphorylase allosterically, by making glycogen phosphorylase more susceptible to the action of another regulatory enzyme. What kind of enzyme is this? Glucose makes the glycogen phosphorylase more susceptible to the action of a protein phosphatase that catalyzes dephosphorylation (and hence deactivation) of the GP. 36) (2 points) Write the general reaction catalyzed by the enzyme involved in glucose regulation of glycogen phosphorylase. You can use P to indicate the protein substrate. P was not a great choice but anyway (the protein is the green P): P-O-PO H 2 O P-OH + HPO 4-2 page 6

7 Glucose coming and going (cont ) (8 points this page) One of the fates of glucose distinct from glycolysis is for the production of ribose. 37) (2 points) What is the name of this pathway? : pentose phosphate pathway, or pentose phophate shunt 38)(4 points) Write the first reaction including the names of products, reactants and cofactor Glu-6P + NADP + 6-phospho-gluconolactone + NADPH 39) (2 points) The second part of this pathway, after ribose production, involves a remarkable collection of interconversions of 4, 5, 7 and 3 carbon sugars. Two types of enzymes are involved in all of those fancy conversions. The enzyme that transfers 2 carbon units is called: transketolase The enzyme that transfers 3 carbon units is called: transaldolase (this is just a little dead space.) page 7

8 Krebsian philosophy (10 points this page) One of ways that Krebs cycle molecules are replenished is by a small group of so-called anaplerotic reactions. One of these mentioned in class that is very important to mammals is catalyzed by phosphoenolpyruvate carboxykinase, called PEP-CK for good reason. 40) (2 points)write the PEP-CK reaction, including all substrates and products. No structures needed. OAA + GTP PEP + GDP + CO 2 41) We have said numerous times in class that you can t use the Krebs cycles for net conversion of acetate into glucose Why is this the case? (One sentence) Every two carbon skeleton that enters the Krebs cycle as acetate is offset by the production of two molecules of C0 2, disallowing net synthesis with acetate. 42) Most of the energy-rich molecules derived from the Krebs cycles are involved in carrying electrons. However, an energy-rich molecule that is not ATP is directly produced by substrate-level phosphoylation. What is this molecule? GTP 43) (2 points) Write the Krebs cycle reaction that is responsible for this substrate level phosphorylation. Include the name of the synthetase (he hinted) that catalyzes it. Names are fine no need for structures, beautiful as the may be. succinyl-coa + GDP succinate + GTP + CoA-SH 44)When an organism does want to use acetate for net synthesis of sugars, an alternate cycle is used. In class we referred to this process as an end run around the Krebs cycle What is the name of this metabolic process? The glyoxalate cycle 45) (2 points) What is the first reaction of this alternate route for acetate carbons? Write the reaction and the name of the enzyme that catalyzes it. Just names, but include all substrates and products. isocitrate lyase isocitrate succinate + glyoxalate 46) Why does this alternate pathway allow net synthesis of things like glucose from acetate? Because there is no release of carbon as CO2, all acetate that enters the pathway can contribute to the net synthesis of molecules such as glucose page 8

9 Cofactor coop (10 points this page) Fill in the blanks Write the cofactor or carrier that best goes with the brief description: 47) Metal ion needed for practically every enzyme that uses ATP or ADP Mg +2 48) Carrier for sugar molecules, used in the conversion of galactose into glucose UDP 49) Undergoes disulfide oxidation/reduction cycle in order to convert pyruvate into acetyl-coa. It acts as a rocker arm in the enzyme we studied lipoic acid or lipoate 50) Used in the carboxylation of many molecules by allowing activation of CO 2 _ biotin 51) (2 pts) Reduced form of the carrier that picks up most of the electrons from Krebs cycle _ NADH _ Molecules at and Exhibition For the structures below, select the letter that corresponds best to the structure. A) NAD B) TPP C) CoA D) biotin E) FAD F) ADP G) PEP H) none 52) D (biotin) 53) A (NAD) page 9 54) E (FAD) 55) B (TPP)

10 Veracity or Fallaciousness (T or F) (12 points this page) 56) F The inner membrane of the mitochondrion is freely permeable to ions 57) T Competitive enzyme inhibitors do not affect the Vmax of a reaction 58) F Krebs cycle occurs in the cytoplasm of mammalian cells. 59) F A positive E' o means a non-spontaneous reaction 60) F An increase in activation energy will change an equilibrium 61) T Regulated metabolic reactions are often far from equilibrium. 62) F All spontaneous reactions give off heat, that is, have a negative delta H 63) F The Krebs cycle only functions in catabolism 64) F ATP is oxidized to ADP and phosphate by water 65) T There are mammalian cells that are almost totally dependent on glycosysis for their energy 66) F Muscles convert pyruvate into ethanol to regenerate NAD + 67) W (word) Nelly is a big fan of glycogen page 10