7.014 Problem Set 2. [substrate] mm Initial reaction velocity* mmol/min

Transcription

1 ame ection Problem et 2 Answers to this problem set are to be turned in at the box outside by 4:00 pm Wednesday, February 20. Problem sets will not be accepted late. olutions will be posted on the web. ( Question 1 The following experimental data were collected during a study of the catalytic activity of an intestinal peptidase capable of hydrolyzing its substrate, the dipeptide glycylglycine: [substrate] mm Initial reaction velocity* mmol/min * Initial velocity is the velocity early in the reaction, before the product has built up enough to slow the reaction rate. a) Plot initial velocity vs [substrate] 1 pring of

2 Question 1, continued b) Using the graph drawn in part (a), estimate K M and V max for the peptidase enzyme. c) ow would doubling the peptidase concentration affect K M? d) ow would doubling the peptidase concentration affect V max? e) ow would doubling the peptidase concentration affect the equilibrium constant (Keq) for the catalyzed reaction? f) ow would doubling the peptidase concentration affect the initial rate of the reaction? g) If you look at the graph you have drawn in part (a), it is linear at low [] and it reaches a plateau at high []. Why does the rate of an enzymecatalyzed reaction plateau at high []? Question 2 The enzyme Phosphofructokinase (PFK) catalyzes the conversion of fructose 6phosphate (F6P) to fructose 1, 6bisphosphate (FBP), in step 3 of glycolysis. fructose 6phosphate phosphofructokinase G o = 3.4 kcal mol fructose 1,6bisphosphate ADP a) What two functions does serve in this coupled reaction? 2 pring of

3 Question 2, continued b) Draw and label an energy diagram for this reaction. Include the relative energy levels of the substrates and the products, the activation energy and the G for the reaction. Reaction Progress In erythrocytes, the following intracellular concentrations of metabolites are found: Metabolite Fructose 6phosphate Fructose 1,6bisphosphate AMP ADP Pi oncentration.014 mm.028 mm 1 mm 0.2 mm 2 mm 1 mm c) What is the free energy change of the phosphofructokinase reaction under these cellular conditions (37 )? how your work. Is the reaction spontaneous under these conditions? Phosphofructokinase (PFK) is the main regulatory enzyme of glycolysis. d) What is the specific signal or molecule(s) to which PFK responds? e) Why does this mechanism of regulation make sense? f) In certain tumor cells, an enzyme called ase becomes abnormally active, resulting in increased hydrolysis of to ADP. What would be the effect on the overall rate of glycolysis in these cells? 3 pring of

4 Question 3 The energy released by hydrolysis can be used to drive unfavorable (endergonic) reactions in the cell. Under ellular onditions hydrolysis Reaction Under tandard onditions (approximate) ADP Pi AMP PPi Go = 7.5 kcal/mol Go = 7.7 kcal/mol G = 12 kcal/mol G = 12.2 kcal/mol For example, fireflies use the energy of hydrolysis of to AMP PP i (pyrophosphate the two terminal phosphate groups of ) to drive the reaction that produces light. ee below: Luciferin (luc) 2 xyluciferin (oxluc) 2 AMP PP i LIGT (one photon per reaction) overall reaction is: rxn 1: : rxn 2: overall rxn: luc 2 luc 2 oxluc light 2 AMP PP i oxluc light 2 AMP PP i In fact, the reaction proceeds by a twostep process. In step 1, reacts with luciferin to produce AMPluciferin PP i. In the second step AMPluciferin reacts with 2 to give AMP 2. This is similar to the glutamine synthetase reaction described in lecture where is used to activate glutamic acid and then the activated glutamic acid reacts to form glutamine. 4 pring of

5 Question 3, continued Given the above G values, and the fact that light production occurs under cellular conditions, what, if anything, can you say about the values of the following G s? ote that, from the information given, you cannot give exact values, only limits or ranges. For example, G rxnx under cellular conditions 27 kcal/mol, G rxny under standard conditions > 0 kcal/mol, or not enough information given. a) G overall rxn under cellular conditions? b) G overall rxn under standard conditions? c) G rxn1 under cellular conditions? Question 4 You notice a slimy patch of goo eating away at your carpet. But wait, your carpet is made of 1950's nylon (called nylon66)! ow can this be? You expect the slimy patch is a colony of rare Archaebacteria, and set out to determine how these bacteria utilize nylon as an energy source. You culture large quantities of the bacteria and painstakingly purify a protein with the ability to degrade nylon. You name the newly discovered enzyme Leggase. ylon is a polymer made up of many repeating subunits (like polysaccharides). It looks like this: The hatched lines at the ends indicate that this same unit is repeated many times in both directions. The arrow points to the bond that is cleaved by the Leggase enzyme. a) If we look at the degradation of nylon in the absence of enzyme the rate at which this bond is cleaved is about 1 per year. In the presence of the enzyme, the rate of cleavage is about 500 bonds/second. What is the increase in the rate of the reaction? 5 pring of

6 Question 4, continued b) You make a solution that is 0.1M nylon polymers. You add Leggase and allow the reaction to reach equilibrium at 25. You determine the concentration of nylon polymers at equilibrium is M. For this problem, assume that 1 molecule of nylon polymer is cleaved into two molecules of cleaved nylon. i) What is the equilibrium constant for the reaction? ii) What is the change in free energy ( G) in kcal/mol? Is this an exergonic or endergonic reaction? c) You realize that this species could be crucial to waste management, but uncontrolled, this bacterium could change life as we know it! You want to be able to control this bacterium, and you discover that a dipeptide, GlyGly is an inhibitor of Leggase. i) Draw the dipeptide GlyGly below. ii) Explain why this dipeptide would inhibit Leggase. d) If nylon66 polymers were aligned in a crystal array, what noncovalent force or bond would dominate the structure? ( 6 pring of

7 tructures of Amino Acids at p 7.0 onpolar 3 3 ALAIE (ala) ILEUIE (ile) 2 3 LEUIE (leu) METIIE (met) 2 3 PEYLALAIE (phe) 2 3 TRYPTPA (trp) 2 3 TYRIE (tyr) VALIE (val) Polar 2 3 ERIE (ser) APARAGIE (asn) 3 3 TREIE (thr) GLUTAMIE (gln) 2 pecial ases: 2 3 YTEIE (cys) 3 GLYIE (gly) PRLIE (pro) harged Acidic Basic ARGIIE (arg) APARTI AID (asp) LYIE (lys) GLUTAMI AID (glu) 2 3 ITIDIE (his) 7 pring of