Name:
2 points
Chem 465
Biochemistry II
Multiple choice (4 points apiece):
1. The anaerobic conversion of 1 mol of glucose to 2 mol of lactate by fermentation is
accompanied by a net gain of:
A)
1 mol of ATP.
B)
1 mol of NADH.
C)
2 mol of ATP.
D)
2 mol of NADH.
E)
none of the above.
2. Which of these cofactors participates directly in most of the oxidation-reduction reactions in
the fermentation of glucose to lactate?
A)
ADP
B)
ATP
C)
FAD/FADH2
D)
Glyceraldehyde 3-phosphate
E)
NAD+/NADH
3. An enzyme used in both glycolysis and gluconeogenesis is:
A)
3-phosphoglycerate kinase.
B)
glucose 6-phosphatase.
C)
hexokinase.
D)
phosphofructokinase-1.
E)
pyruvate kinase.
4. Gluconeogenesis must use "bypass reactions" to circumvent three reactions in the glycolytic
pathway that are highly exergonic and essentially irreversible. Reactions carried out by which
three of the enzymes listed must be bypassed in the gluconeogenic pathway?
1)
Hexokinase
2)
Phosphoglycerate kinase
3)
Phosphofructokinase-1
4)
Pyruvate kinase
5)
Triosephosphate isomerase
A)
B)
C)
D)
E)
1, 2, 3
1, 2, 4
1, 4, 5
1, 3, 4
2, 3, 4
5. Cellular isozymes of pyruvate kinase are allosterically inhibited by:
A)
high concentrations of AMP.
B)
high concentrations of ATP.
C)
high concentrations of citrate.
D)
low concentrations of acetyl-CoA.
E)
low concentrations of ATP.
6. Which of the below is not required for the oxidative decarboxylation of pyruvate to form
acetyl-CoA?
A)
ATP
B)
CoA-SH
C)
FAD
D)
Lipoic acid
E)
NAD+
7. Which one of the following enzymatic activities would be decreased by thiamine deficiency?
A)
Fumarase
B)
Isocitrate dehydrogenase
C)
Malate dehydrogenase
D)
Succinate dehydrogenase
E)
á-Ketoglutarate dehydrogenase complex
Longer questions - 15 points each - Choose any 4!
8. What is Fructose 2,6-bisphosphate? Where does it come from? And how is it important in the
regulation of glycolysis and gluconeogenesis? (Include as many details as possible)
Fructose 2,6 biphosphate is used primarily as an allosteric effector of
the enzymes phosphofructokinase-1 and fructose 1,6 biphosphatase-1.
Fructose 2,6-biphosphate is made from Fructose-6-P by the enzyme
Phosphofructokinase-2 and is converted back to F-6-P by the enzyme
fructose 2,6-biphosphatse-2. Both of these enzymatic activities are
combined in one bifuctional protein that toggles between on activity
and the other based on the phosphorylation of the enzyme. The complete control cycle looks like
this.
High Blood sugar
insulin released
phosphoprotein phosphatase stimulated
protein is dephosphorylated and changes to PFK-2 active form
Fructose 6-P is changed to Fructose 2,6-biphosphate
[Fructose 2,6-biphosphate]8
This activates Phosphofructokinase - 1 and stimulates glycolysis to make more
energy and use up glucose
This also inhibits fructose 1,6 biphosphatase so gluconeogenesis is slowed so no
additional glucose is made
Low Blood sugar
Glucagon released
Adenylate cyclase makes cAMP
cAMP dependent protein kinase phosphorylates enzyme and changes to FBPase-2 Active
form
[Fructose 2,6-biphosphate]9
Phosphofructokinase - 1 no longer activated so glycolysis is slowed and use of
glucose is reduced.
1,6 biphosphatase is no longer inhibited so gluconeogenesis is can start up and
additional glucose is made.
9. If I have glucose labeled with 13C in the 2 position (see diagram below) where does the 13C
label end up in the following compounds? (Draw the structure of the compound and indicate the
position of the label) Assume all reactions are going in the normal glycolytic direction, and no
gluconeogenesis is taking place.
Fructose 1,6-bisphosphate
Glyceraldehyde 3-phosphate
No label from initial aldolase reaction
But after triose phosphate isomerase rxn
½ of the molecules will be
Dihydroxyacetone phosphate
Phosphoenolpyruvate
½ of the molecules
Pyruvate
½ of the molecules
Lactate (low oxygen)
½ of molecules
(Page left blank so you can sketch out the glycolytic pathway to get your answers)
10. I am going to add glutamic acid labeled in the ä position with 13C (See diagram below) to a
cell that is using the TCA cycle to generate energy. Name, and draw the structure of the first
intermediate in the TCA cycle in which this label will appear. Indicate which C in this
intermediate will carry the 13C label. Initially glutamate will enter the TCA as á-ketoglutarate as labeled below:
Now draw the structures of these other TCA intermediates, and indicate where this label will
appear in these compounds
Succinyl-CoA
Fumarate
(Note: due to symmetry both ends equivalent)
Oxaloacetate
Citrate
Note: due to symmetry in Succinate and Fumarate a single molecule of oxaloacetate will only
have a label at one COO- or the other, but not both. Similarly a single molecule of citrate will
have label at only one of the two positions indicated.
Isocitrate
á-Ketoglutarate
Again only one of the labeled positions are labeled in any single isocitrate molecule.
Only ½ of the á-ketoglutarate molecules will be carrying a label after one turn of the TCA cycle.
(Page left blank so you can sketch out the TCA pathway to get your answers)
11. What is biotin? What is it used for biochemically? What enzymes have we studied that use
biotin as an integral part of their mechanism.
Biotin is a coenzyme or cofactor that carries activated CO2 in the active site of an enzyme. The
structure of biotin is shown below, but you were not required to know this structure.
We saw this cofactor in the enzyme pyruvate carboxylase which adds
CO2 to pyruvate to make oxaloacetate. This oxaloacetate can either be
used to replace oxaloacetate that hs been removed from the TCA cycle,
or it can be used for the first step in gluconeogenesis. While Biotin is
technically an vitamin because you cannot synthesize it, the bacteria in
your gut can make this factor, so it is very difficult to develop a biotin
deficiency. Biotin deficiencies can occur, however, in people who eat
raw eggs because uncooked eggs contain the protein avidin which binds
biotin so it cannot be absorbed in the gut.
12. Compare and contrast the structure, mechanism, cofactors used, and regulation of pyruvate
dehydrogenase complex and á-ketoglutarate dehydrogenase complex.
Pyruvate dehydrogenase catalyzes the reaction Pyruvate + CoASH 6AcetylCoA + NADH + H+
á-ketoglutarate dehydrogenase catalyzes the reaction á-ketoglutarate + CoASH 6SuccinylCoA +
NADH + H+.
Pyruvate dehydrogenase and á-ketoglutarate dehydrogenase are very similar in structure and
mechanism. Both are large enzyme complexes that contain multiple copies of three major
proteins usually called simply E1, E2, and E3. Both enzyme complexes also utilize the cofactors
thiamine pyrophosphate, lippoate and FAD, ad well as the substrate CoenzymeA.
In pyruvate dehydrogenase the E1 protein is actually the pyruvate dehydrogenase part of the
complex. It uses TPP to cleave the terminal CO2 from pyruvate and transfers the remaining part
of the pyruvate to E2 (Dihydrolipoyl transacetylase) where this acetate group is temporarily
attached to lippoate. The acetate is then transferred to free CoASH to make acetyl CoA which
diffuses away from the enzyme complex. When this transfer occurs the lipoate is left in a fully
reduced form that must be regenerated before the enzyme can work again. This oxidation is
performed by the E3 enzyme, dihydrolipoyl dehydrogenase, which transfers the protons from the
reduced lipoate to NAD+ to make NADH.
The E3 enzyme is virtually identical in both complexes because it does exactly the same
biochemical reaction. The E1 enzyme is just a bit different in the two complexes because it must
bind different substrates (pyruvate or á-ketoglutarate). The E2 complexes are close in structure
because they also do the same chemistry, but in they vary slightly because different one creates
acetyl CoA and the other makes sussinyl-CoA.
The allosteric control of pyruvate dehydrogenase is a bit more complicated because it is the first
step in the TCA pathway. Pyruvate dehydrogenase is stimulated AMP, CoA, NAD+, and Ca2+
and inhibited by ATP, acetyl-CoA, NADH. The á-ketoglutarate dehydrogenase control is
simpler because it is deeper inside the TCA cycle. It is only stimulated by Ca2+ and inhibited by
succinyl-CoA and NADH.
1: C
2: E
3: A
4: D
5: B
6: A
7: E