Exam 3, BICH 410, Section 503 (MWF 3-3:50), Friday, November 20, 1998

Write your name on each page. Write concise answers to demonstrate effectively your mastery of the subject material. Show your work in order to receive partial credit where applicable.

1. (10 pts) Match the cofactors below with their roles in the pyruvate dehydrogenase complex reaction.
Cofactors
Roles (Answers included)
_B__ Attacks and attaches to the central carbon in pyruvate
__F__ Initial electron acceptor in oxidation of pyruvate
__A__ Oxidizes FADH2
__E__ Accepts the acetyl group from reduced lipoic acid
__C__ Oxidizes the reduced form of lipoic acid

2. (15 pts) Starting with the hormone epinephrine, diagram the cascade of steps in the signal transduction pathway in a muscle cell ending with the phosphorolysis of glycogen to glucose-1-phosphate. Include in your answer the role of calcium in this process.

Answer:
1) Epinephrine binds receptor
2) GTP binds G protein
3) Adenyl cyclase activated to produce cAMP from ATP
4) cAMP activates protein kinase A
5) Protein kinase A + ATP phosphorylates phosphorylase b kinase
6) Ca++-calmodulin also needed to activate phosphorylase kinase
7) Phosphorylase b kinase + ATP phosphorylates phosphorylase b to convert to phosphorylase a
8) Phosphorylase a catalyzes phosphorolysis of glycogen to glucose-1-phosphate

3. (15 pts) For the case of a competitive inhibitor that binds to an enzyme displaying Michaelis-Menten kinetics, answer the following questions:
A) Write the binding equilibria for the interaction of the enzyme (E) with substrate (S) and/or inhibitor (I), including the formation of the enzyme-substrate complex and product (P).
Answer: E + S <-> ES -> E + P
For inhibitor binding, E + I <-> EI; equilibrium described by KI

B) Draw velocity vs. [S] curves for the enzyme-catalyzed reaction with and without addition of inhibitor. Label the axes.
C) Draw Lineweaver-Burk plots for the enzyme-catalyzed reaction with and without addition of inhibitor. Label the axes and demonstrate the significance of the extrapolation of these plots with the x- and y-axes.
D) Demonstrate how you would calculate KI, the dissociation constant for binding of the inhibitor to the enzyme.
Answers for B, C, and D in book, page 385

4. (16 pts) Starting with glucose radiolabeled at both carbons 1 and 2, draw the structures of the following metabolites, specifying which carbons are derived from the radiolabeled glucose via glycolysis and/or the citric acid cycle. Clearly denote the labeled carbon atoms in each metabolite with either a 1 (from glucose C-1) or a 2 (from glucose C-2).
A) glucose-6-phosphate
B) glyceraldehyde-3-phosphate
C) pyruvate
D) isocitrate
Answers on key

5) (8 pts) Draw the structures of the following coenzyme-acetyl intermediates involved in the reactions catalyzed by the pyruvate dehydrogenase complex:
A) hydroxyethyl-thiamine pyrophosphate "business part" (active acetaldehyde):
B) acetyl group attached to "business part" of lipoic acid:
Answers on key

6) (8 pts) In the vertebrate liver, fructose can be metabolized starting with a reaction catalyzed by fructokinase. From the following list of compounds and cofactors, select those that are involved in the production of pyruvate in this pathway.

phosphofructokinase, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate dehydrogenase, NAD+, aldolase B, lipoic acid, fructose-1-phosphate, acetyl-CoA, glyceraldehyde, UDP-glucose, phosphoenolpyruvate, CO2, phosphoglycerate kinase

Answer: fructose-1-phosphate, aldolase B, dihydroxyacetone phosphate, glyceraldehyde, glyceraldehyde-3-phosphate dehydrogenase, NAD+, phosphoglycerate kinase, phosphoenolpyruvate

7) (8 pts) Draw velocity vs. substrate curves (on the same axes) demonstrating allosteric regulation of aspartate transcarbamoylase (ATCase) in the presence of i) no regulator, ii) CTP, and iii) ATP. Show on the graphs how the K0.5 values can be determined for each case. What is the biochemical regulatory sense for the CTP effect on this enzyme?
Answer: see key for graph; regulatory sense: ATCase catalyzes an early step in pyrimidine nucleotide biosynthesis. If [CTP] is higher than needed, it feedback inhibits ATCase to reduce further biosynthesis of pyrimidines.

8) (20 pts) Fill in the blanks:
A) two positive regulators for phosphofructokinase
Answer: ADP or AMP (or low energy charge); fructose-2,6-bisphosphate
B) two glycolytic enzymes that catalyze isomerization of a ketose to an aldose, or vice versa
Answer: phosphoglucoisomerase, triosephosphate isomerase
C) reaction catalyzed by glycerokinase (either names or structures OK)
Answer: Glycerol + ATP -> glycerol-3-phosphate + ADP
D) two negative regulators for pyruvate kinase
Answer: ATP (high energy charge), acetyl-CoA (or fatty acids)
E) glycolytic enzyme inhibited by iodoacetate
Answer: glyceraldehyde-3-phosphate dehydrogenase
F) names of two zymogens activated by trypsin
Answer: chymotrypsinogen, trypsinogen, proelastase, procarboxypeptidase
G) name of enzyme missing in lactose-intolerant individuals
Answer: lactase
name of enzyme found in saliva that hydrolyzes alpha(1->4) linkage of starch
Answer: alpha-amylase
H) names of three reactions in glycolysis that are irreversible (deltaG' << 0) (write names of enzymes)
Answer: hexokinase, phosphofructokinase, pyruvate kinase
I) names of two glycolytic intemediates (not enzymes) that donate phosphate for substrate-level phosphorylation reactions
Answer: 1,3-bisphosphoglycerate, phosphoenolpyruvate