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2. Lactate dehydrogenase (LDH) catalyzes the reaction Ο ()) 0 NADH + H* NAD+ C=0 HO-C-H CH₁t Pyruvate lactate dehydrogenase CHa L-Lactate AG 25.1 kJ/mol which represents one of the metabolic fates of pyruvate, the end product of glycolysis. The positioning of the sub-strate pyruvate in the active site of lactate dehydrogenase is shown on the right. NADH (nicotinamide adenine dinu- cleotide) is a cofactor in the reaction and provides a hydride anion H (highlighted with light blue) through direct transfer to reduce the carbonyl group of pyruvate. (a) ( Gln 102 Arg109 NH2 NH Thr246 H₂C-C-OH HN NH H CHS H H H. His 195 NH Pyruvate N-(NADH) H H CH3 H HN H NH H₂C-C-CH₂ Пle250 Asp168 NH Arg171 ) Compare the mechanism of the LDH reaction, as implied by the diagram above, to that of a-chymotrypsin with respect to the oxyanion hole, conversion of the substrate carbonyl group having sp² hybridization to sp³ hybridization, the attacking nucleophile, and residues achoring the substrate in the active site. Oxyanion hole α-chymotrypsin LDH sp² to sp³ hybridization Attacking nucleophile Substrate anchoring (b) A mutant of LDH in which Arg171 is substituted by lysine shows greatly reduced activity. Why might this be surprising and what is the likely reason for the unexpected reduction in activity? (c) ) Substitution of Arg for Gln102 produced an enzyme that preferentially catalyzed reduction of oxaloacetate instead of pyruvate. This reaction is written below as COO NADH + H+ NAD* O=C CH2 coo Oxaloacetate ၄၀၀- HO-C-H CH₂ coo- L-Malate Explain the structural basis of this reaction according to the factors listed in part (a). The mutant Gln- 102Arg enzyme no longer would reduce pyruvate. Suggest how the active site may have become distorted, preventing it from binding pyruvate in a catalytically competent conformation.

2. Lactate dehydrogenase (LDH) catalyzes the reaction Ο ()) 0 NADH + H* NAD+ C=0 HO-C-H CH₁t Pyruvate lactate dehydrogenase CHa L-Lactate AG 25.1 kJ/mol which represents one of the metabolic fates of pyruvate, the end product of glycolysis. The positioning of the sub-strate pyruvate in the active site of lactate dehydrogenase is shown on the right. NADH (nicotinamide adenine dinu- cleotide) is a cofactor in the reaction and provides a hydride anion H (highlighted with light blue) through direct transfer to reduce the carbonyl group of pyruvate. (a) ( Gln 102 Arg109 NH2 NH Thr246 H₂C-C-OH HN NH H CHS H H H. His 195 NH Pyruvate N-(NADH) H H CH3 H HN H NH H₂C-C-CH₂ Пle250 Asp168 NH Arg171 ) Compare the mechanism of the LDH reaction, as implied by the diagram above, to that of a-chymotrypsin with respect to the oxyanion hole, conversion of the substrate carbonyl group having sp² hybridization to sp³ hybridization, the attacking nucleophile, and residues achoring the substrate in the active site. Oxyanion hole α-chymotrypsin LDH sp² to sp³ hybridization Attacking nucleophile Substrate anchoring (b) A mutant of LDH in which Arg171 is substituted by lysine shows greatly reduced activity. Why might this be surprising and what is the likely reason for the unexpected reduction in activity? (c) ) Substitution of Arg for Gln102 produced an enzyme that preferentially catalyzed reduction of oxaloacetate instead of pyruvate. This reaction is written below as COO NADH + H+ NAD* O=C CH2 coo Oxaloacetate ၄၀၀- HO-C-H CH₂ coo- L-Malate Explain the structural basis of this reaction according to the factors listed in part (a). The mutant Gln- 102Arg enzyme no longer would reduce pyruvate. Suggest how the active site may have become distorted, preventing it from binding pyruvate in a catalytically competent conformation.

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
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2. Lactate dehydrogenase (LDH) catalyzes the reaction Ο ()) 0 NADH + H* NAD+ C=0 HO-C-H CH₁t Pyruvate lactate dehydrogenase CHa L-Lactate AG 25.1 kJ/mol which represents one of the metabolic fates of pyruvate, the end product of glycolysis. The positioning of the sub-strate pyruvate in the active site of lactate dehydrogenase is shown on the right. NADH (nicotinamide adenine dinu- cleotide) is a cofactor in the reaction and provides a hydride anion H (highlighted with light blue) through direct transfer to reduce the carbonyl group of pyruvate. (a) ( Gln 102 Arg109 NH2 NH Thr246 H₂C-C-OH HN NH H CHS H H H. His 195 NH Pyruvate N-(NADH) H H CH3 H HN H NH H₂C-C-CH₂ Пle250 Asp168 NH Arg171 ) Compare the mechanism of the LDH reaction, as implied by the diagram above, to that of a-chymotrypsin with respect to the oxyanion hole, conversion of the substrate carbonyl group having sp² hybridization to sp³ hybridization, the attacking nucleophile, and residues achoring the substrate in the active site. Oxyanion hole α-chymotrypsin LDH sp² to sp³ hybridization Attacking nucleophile Substrate anchoring (b) A mutant of LDH in which Arg171 is substituted by lysine shows greatly reduced activity. Why might this be surprising and what is the likely reason for the unexpected reduction in activity? (c) ) Substitution of Arg for Gln102 produced an enzyme that preferentially catalyzed reduction of oxaloacetate instead of pyruvate. This reaction is written below as COO NADH + H+ NAD* O=C CH2 coo Oxaloacetate ၄၀၀- HO-C-H CH₂ coo- L-Malate Explain the structural basis of this reaction according to the factors listed in part (a). The mutant Gln- 102Arg enzyme no longer would reduce pyruvate. Suggest how the active site may have become distorted, preventing it from binding pyruvate in a catalytically competent conformation.
Transcribed Image Text:2. Lactate dehydrogenase (LDH) catalyzes the reaction Ο ()) 0 NADH + H* NAD+ C=0 HO-C-H CH₁t Pyruvate lactate dehydrogenase CHa L-Lactate AG 25.1 kJ/mol which represents one of the metabolic fates of pyruvate, the end product of glycolysis. The positioning of the sub-strate pyruvate in the active site of lactate dehydrogenase is shown on the right. NADH (nicotinamide adenine dinu- cleotide) is a cofactor in the reaction and provides a hydride anion H (highlighted with light blue) through direct transfer to reduce the carbonyl group of pyruvate. (a) ( Gln 102 Arg109 NH2 NH Thr246 H₂C-C-OH HN NH H CHS H H H. His 195 NH Pyruvate N-(NADH) H H CH3 H HN H NH H₂C-C-CH₂ Пle250 Asp168 NH Arg171 ) Compare the mechanism of the LDH reaction, as implied by the diagram above, to that of a-chymotrypsin with respect to the oxyanion hole, conversion of the substrate carbonyl group having sp² hybridization to sp³ hybridization, the attacking nucleophile, and residues achoring the substrate in the active site. Oxyanion hole α-chymotrypsin LDH sp² to sp³ hybridization Attacking nucleophile Substrate anchoring (b) A mutant of LDH in which Arg171 is substituted by lysine shows greatly reduced activity. Why might this be surprising and what is the likely reason for the unexpected reduction in activity? (c) ) Substitution of Arg for Gln102 produced an enzyme that preferentially catalyzed reduction of oxaloacetate instead of pyruvate. This reaction is written below as COO NADH + H+ NAD* O=C CH2 coo Oxaloacetate ၄၀၀- HO-C-H CH₂ coo- L-Malate Explain the structural basis of this reaction according to the factors listed in part (a). The mutant Gln- 102Arg enzyme no longer would reduce pyruvate. Suggest how the active site may have become distorted, preventing it from binding pyruvate in a catalytically competent conformation.
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