BIOCHEMISTRY - L. Stryer - 1984

VOLUME 2

ANSWERS TO QUESTIONS AND PROBLEMS

Chapter 11

1. Reactions (a) and (c) are favored to the left; reactions (b) and (d) are favored to the right.

2. In no way (they cannot be used for this purpose).

3. a) ∆G⁰' = + 7.5 kcal/mol and K_éq = 3.16 • 10- ⁶. b) 3.16 • 10⁴.

4. ∆G^0, = 1.7 kcal/mol. The equilibrium ratio is 17.8.

5. a) + 0.2 kcal/mol. b) - 7.8 kcal/mol. Hydrolysis of PPi drives the reaction toward the Formation of Acetyl-CoA.

6. a) ∆G₀ = 2.303 RT pK. b) - 6.53 kcal/mol at 25C.

7. The ADP moiety (or a closely related derivative in the case of CoA).

8. The activated form of sulfate in most organisms is 3'-phosphoadenosine-5'-phosphosulfate.

Chapter 12

1. a) Aldose-ketose; b) epimers; c) aldose-ketose; d) anomers; e) aldose-ketose; f) epimers.

2. The methyl carbon of Pyruvate is labeled with 14_C

3. a) ∆G⁰’ is -29.5 kcal/mol for the reaction

Glucose + 2Pi + 2ADP → 2 Lactate + 2ATP. b) ∆G⁰' = -27.2 kcal/mol.

4. 3.06 • 10^-5.

5. The equilibrium concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, and glyceraldehyde 3-phosphate are 7.76 • 10^-4, 2.24 • 10^-4, and 2.24 • 10^-4 M, respectively.

6. All three carbon atoms of 2,3-BPG are labeled with ¹⁴C. The phosphorus atom attached to the C-2 hydroxyl group is labeled with ³²P.

7. Hexokinase has low ATPase activity in the absence of sugar because it is in a catalytically inactive conformation (Section 12.12). The addition of xylose closes the cleft between the two lobes of the enzyme. However, xylose lacks the hydroxymethyl group at C-6 and thus cannot be phosphorylated. Instead, a Water molecule acts as the acceptor of the phosphoryl group transferred from ATP, occupying the site normally filled by the C-6 hydroxymethyl group.

8. X-ray crystallographic studies by Winn S. I., Watson H. C., Harkins R. N., Fothergill L. A., Biochem. Soc. Trans., 5, 657-659 (1978) show that 3-phosphoglycerate binds to the Active Site of the enzyme, in which Histidine-184 is phosphorylated and histidine-8 is free. Phosphohistidine-184 transfers its phosphoryl group to the substrate, yielding 2,3-bisphosphoglycerate. The phosphoryl group at the 3-position of this intermediate is then transferred to histidine-8, forming 2-phosphoglycerate. The original form of the enzyme is regenerated by the subsequent transfer of the phosphoryl group from histidine-8 to histidine-184. Note the similarity of this catalytic mechanism to that of bisphosphoglycerate mutase (Section 12.17).

Chapter 13

1. a) After one turn of The Citric Acid Cycle, the label appears in C-2 and C-3 of oxaloacetate.

b) After one turn of The Citric Acid cycle, the label appears in C-1 and C-4 of oxaloacetate.

c) The label appears in CO₂ during The formation of acetyl-CoA from pyruvate.

d, e) The Fate of the label is the same as in case 'a'.

2. No, because two carbon atoms are lost in the cycle during the two decarboxylation steps. Therefore, oxaloacetate cannot accumulate under these conditions.

3. 0.90, 0.03, and 0.07.

4. -9.8 kcal/mol.

5. The stereospecificity of glyceraldehyde-3-phosphate dehydrogenase with respect to the coenzyme is opposite to that of Alcohol dehydrogenase (type B and type A, respectively).

6. Thiamine thiazolone pyrophosphate is an analog of the Transition State. The sulfur-containing ring of this analog carries no charge, and therefore it is very similar to the intermediate state of the normal coenzyme in thiamine-catalyzed reactions (i.e., to the uncharged Resonance form of hydroxyethyl-TPP, Sec. 13.10).

7. For oxaloacetate to be formed, The ratio of malate to oxaloacetate must exceed 1.75 • 10⁴.

Chapter 14

1. a) 15; b) 2; c) 38; d) 16; e) 36; and f) 19.

2. a) ∆E'o for this reaction is +1.05 V, and ∆G⁰' is -48.4 kcal/mol.

2Г-SH + 1/2О₂ ⇄ Г-SS-Г + Н₂О.

b) ∆Eô = +0.09 V, and ∆G⁰' is -4.15 kcal/mol.

3. a) Blocks Electron Transport and the proton pump at site 3.

b) Blocks electron transport and ATP synthesis by inhibiting ATP and ADP exchange across The inner mitochondrial membrane.

c) Blocks electron transport and the proton pump at site 1.

d) Blocks ATP synthesis by disrupting the proton gradient without inhibiting electron transport.

e) Blocks electron transport and the proton pump at site 3.

f) Blocks electron transport and the proton pump at site 2.

4. Oligomycin inhibits ATP synthesis by preventing the utilization of the proton gradient. It does not block electron transport.

5. ∆G⁰' is +16.1 kcal/mol for oxidation with NAD+ and +1.4 kcal/mol for oxidation with FAD. The reduction of succinate by NAD⁺ is thermodynamically impossible.

6. Cyanide can have a lethal effect by binding to the ferric form of cytochrome (a + a₃) and thereby inhibiting Oxidative Phosphorylation. Nitrite converts ferrohemoglobin to ferrihemoglobin, which also binds cyanide. Thus, ferrihemoglobin competes with cytochrome (a + a₃) for cyanide. This competition has a therapeutic effect because The amount of ferrihemoglobin that can be formed without impairing Oxygen transport far exceeds the amount of cytochrome (a⁺+ a3).

7. The available Free energy resulting from the translocation of 2, 3, and 4 protons is -9.23, -13.8, and -18.5 kcal, respectively. The free energy consumed in the synthesis of 1 mol of ATP under standard conditions is 7.3 kcal. Consequently, the remaining Free energy of -1.93, -6.5, and -11.2 kcal can drive ATP synthesis until the [ATP]/[ADP][Pi] ratio reaches 26.2, 6.51 • 104, and 1.62 • 10⁸, respectively. Suspensions of isolated Cell/35.html">Mitochondria synthesize ATP until this ratio exceeds 104. This implies that at least three protons are translocated per mole of ATP synthesized.

Chapter 15

1. a) 5 Glucose-6-phosphate + ATP → 6 Ribose-5-phosphate + ADP + H+.

b) Glucose 6-phosphate + 12NADP⁺ + 7H₂O → 6CO₂ + 12NADPH + 12H⁺ + Pi.

2. The label appears at C-5 of ribulose 5-phosphate.

3. Oxidative Decarboxylation of isocitrate to α-ketoglutarate. In both reactions, a β-keto acid is formed as an intermediate.

4. In fructose 6-phosphate, the label is found at C-1 and C-3, whereas in erythrose 4-phosphate, the label is absent.

5. Reactions b and e are blocked.

6. Form a Schiff base between the ketose substrate and transaldolase, reduce it with tritiated NaBH₄, and analyze the labeled enzyme by fingerprinting.

Chapter 16

1. Galactose + ATP + UTP + H₂O + Glycogen n → Glycogen_n+1 + ADP + UDP + 2Pi + H⁺.

2. Fructose + 2 ATP + 2H₂O → Glucose + 2ADP + 2Pi.

3. Branching enzyme deficiency is present.

4. In Von Gierke's disease, the glucose 6-phosphate concentration is elevated. Consequently, the phosphorylated D-form of Glycogen synthase is active.

5. Glucose is an allosteric inhibitor of phosphorylase a. Consequently, crystals grown in its presence are in the T-state. The addition of glucose 1-phosphate, the substrate of phosphorylase, shifts the R ⇄ T equilibrium toward the R-state. The conformational differences between these states are large enough to cause the crystals to shatter unless they are stabilized by chemical cross-linking. Crystal shattering induced by an allosteric transition was first described by Haurowitz for the oxygenation of deoxyhemoglobin crystals.

6. Hers [Hers, H. G., Ann. Rev. Biochem., 45, 167 (1976)] suggested that The kinetics of these reactions account for a lag period in the dephosphorylation of the B subunit, thereby allowing glycogen breakdown to occur before phosphorylase kinase is inactivated by its phosphatase.

Chapter 17

1. a) Glycerol + 2NAD⁺+ Pi + ADP → Pyruvate + ATP + H₂O + 2 NADH + H⁺.

b) Glycerol kinase and glycerol phosphate dehydrogenase.

2. Stearate + ATP + 131/2 H₂O + 8 FAD + 8 NAD⁺ → 4 • 1/2Acetoacetate + 12 • 1/2H+ + 8FADH₂ + 8FADH + AMP + 2P.

3. a) Oxidation in mitochondria, synthesis in the Cytosol.

b) Acetyl-CoA for oxidation, acyl carrier protein for synthesis.

c) FAD and NAD⁺ for oxidation, NADPH for synthesis.

d) L-isomer of 3-hydroxyacyl-CoA in oxidation, D-isomer in synthesis.

e) From carboxyl to methyl in oxidation, from methyl to carboxyl in synthesis.

f) Fatty acid synthesis Enzymes, but not oxidation enzymes, are organized into a multienzyme complex.

4. a) Palmitoleate; b) linoleate; c) linoleate; d) oleate; e) oleate; and f) linolenate.

5. C-1 has higher radioactivity (see the Structure/133.html">Discussion of this experimental approach for determining the direction of Polypeptide chain synthesis in Sec. 27.11).

6. a) Yes.

2 Acetyl-CoA + 3H₂O + FAD + 2NAD⁺ → Oxaloacetate + 2CoA + FADH₂ + 2NADH + 4H⁺.

c) Yes, because glucose can be synthesized from oxaloacetate via The Gluconeogenesis pathway.

Chapter 18

1. a) Pyruvate; b) oxaloacetate; c) α-oxoglutarate; d) α-oxoisocaproate; e) phenylpyruvate and f) hydroxyphenylpyruvate.

2. Aspartate + α-Oxoglutarate + GTP + ATP + 2 H₂O + NADH + H⁺ → 1/2 Glucose + Glutamate + CO₂ + ADP + GDP + NAD⁺ + 2Pi.

3. Aspartate + CO₂ + NH +4 + 3 ATP + NAD⁺ +-4 H₂O → Oxaloacetate + Urea + 2 ADP + 4Pi + AMP + NADH + H⁺.

4. a) Label the ¹⁴C carbon atom of the methyl group of L-methylmalonyl-CoA. Determine the localization of ¹⁴C in succinyl-CoA. The migrating group is the one bonded to the labeled carbon atom.

b) Label the ¹⁴C carbon atom of the methyl group of L-methylmalonyl-CoA and label the ³⁵S sulfur atom of its CoA component. The transfer of the -CO—S—CoA group will be intramolecular if succinyl-CoA is found to contain both ¹⁴C and ³⁵S.

c) The proton of the methyl group of L-methylmalonyl-CoA is directly transferred to the adjacent carbon atom.

5. Thiamine pyrophosphate.

Chapter 19

1. ∆E0 = + 0.28 V and ∆G⁰' = — 12.9 kcal/mol.

2. Aldolase is involved in The Calvin Cycle, whereas transaldolase is involved in the Pentose Phosphate Pathway.

3. The concentration of 3-phosphoglycerate will increase, and the concentration of ribulose 1,5-bisphosphate will decrease.

4. The concentration of 3-phosphoglycerate will decrease, and the concentration of ribulose 1,5-bisphosphate will increase.

5. Phycoerythrin and phycocyanin serve as antenna molecules. They absorb light in the spectral region where chlorophyll a has low absorption, and then transfer their excitation energy to chlorophyll a.

6. a) This is an expression of a key aspect of Photosynthesis: water is split by light. The oxygen released during photosynthesis comes from water.

b) Van Niel's equation for Respiration reflects the fact that the combustion of glucose requires the input of six H₂O molecules. See the interesting discussion on The Nature of cellular respiration.

7. The addition of pyridine increases the proton-accumulating capacity of the thylakoid lumen. Thus, a larger number of protons can flow in the dark through the ATP-synthesizing complex.

8. Dichlorophenyldimethylurea (DCMU) inhibits electron transfer between coenzyme Q and plastoquinone at the site linking Photosystems II and I. O₂ evolution can occur in the presence of DCMU, provided an artificial electron acceptor, such as ferricyanide, is available to accept electrons from coenzyme Q.

Chapter 20

1. Glycerol + 4 ATP 4- 3 Fatty acid + 4 H₂O → Triacylglycerol + ADP + 3AMP + 7P¡ + 4H⁺.

2. Glycerol + 3 ATP + 2 Fatty acid + 2 H₂O + CTP + Serine → Phosphatidylserine + CMP + ADP + 2AMP + 6P¡ + 3H⁺

3. a) CDP-diacylglycerol; b) CDP-ethanolamine; c) Acyl-CoA; d) CDP-Choline; e) UDP-glucose or UDP-galactose; f) UDP-galactose; g) geranyl pyrophosphate.

4. a) and b) The label will not be detected anywhere, as it is lost as CO₂.

Chapter 21

1. Glucose + 2ADP + 2 P¡ + 2NAD⁺+ 2 Glutamate → 2 Alanine + 2α-Ketoglutarate + 2 ATP + + 2NADH + H⁺.

2. N₂→ NH4 → Glutamate → Serine → Glycine → δ-Aminolevulinate → Porphobilinogen → Heme.

3. a) Tetrahydrofolate; b) Tetrahydrofolate; c) N⁵-methyltetrahydrofolate.

4. The reaction intermediate is likely γ-glutamyl phosphate.

5. Administration of glycine leads to the formation of isovalerylglycine. Unlike valeric acid, this compound is water-soluble and rapidly excreted by the Kidneys.

6. The pattern of H–D exchange indicates that diimide is formed as an intermediate.

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7. They carry out Nitrogen Fixation. Due to the absence of Photosystem II, an oxygen-free environment is created within them. Recall that Nitrogenase is very rapidly inactivated by oxygen.

Chapter 22

1. Glucose + 2 ATP + 2NADP⁺ + H₂O → PRPP + CO₂ + ADP + AMP + 2NADPH + H⁺.

2. Glutamine + Aspartate + CO₂ + 2 ATP + NAD⁺ → Orotate + 2ADP + 2 Pi + Glutamate + NADH + H⁺.

3. a, c, d, and e) PRPP; b) carbamoyl phosphate.

4. PRPP and formylglycinamide ribonucleotide.

5. dUMP + Serine + NADPH + H⁺ → dTMP + NADP⁺ + Glycine.

6. This creates a deficiency of N10-formyltetrahydrofolate. Sulfanilamide inhibits Folic acid synthesis because it is an analog of one of its precursors, p-aminobenzoic acid.

7. PRPP is an activated intermediate in the following biosynthetic pathways: a) in the synthesis of phosphoribosylamine during de novo purine synthesis; b) in the Synthesis of purine NUCLEOTIDES from free bases (salvage pathway); c) in the synthesis of orotidylic acid during pyrimidine synthesis; d) in the synthesis of nicotinic acid ribonucleotide; e) in the formation of phosphoribosyl-ATP during histidine synthesis; f) in the formation of phosphoribosyl anthranilate during Tryptophan synthesis.

8. Most likely, ammonia is cleaved from glutamine As a result of the catalytic action of the small subunit. The newly formed ammonia then reacts with an activated form of CO₂ generated by the large subunit. The fact that the subunit possesses bicarbonate-dependent ATPase activity suggests that the activated form is carboxyphosphate. The reaction of this mixed carbonic-phosphoric anhydride with NH₃ yields carbamate, which then reacts with ATP to produce carbamoyl phosphate.