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Role of the S128, H186, and N187 triad in substrate binding and decarboxylation in the sheep liver 6-phosphogluconate dehydrogenase reaction.
Biochemistry. 2006 Oct 24; 45(42):12680-6.B

Abstract

Crystal structures of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver indicate that S128 and N187 are within hydrogen-bonding distance of 6PG in the E:6PG binary complex and NADPH in the E:NADPH binary complex. In addition, H186 is also within hydrogen-bonding distance of NADPH in the E:NADPH binary complex, while in the E:6PG binary complex it is within hydrogen-bonding distance of S128 and close to N187. The structures suggest that this triad of residues may play a dual role during the catalytic reaction. Site-directed mutagenesis has been performed to mutate each of the three residues to alanine. All mutant enzymes exhibit a decrease in V/E(t) (the turnover number), ranging from 7- to 67-fold. An increase in the Km for 6PG (K(6PG)) was observed for S128A and H187A mutant enzymes, while for the H186A mutation, K(6PG) is decreased by a factor of 2. K(NADP) remains the same as the wild type enzyme for the S128A and H186A mutant enzyme, while it increases by 6-fold in the N187A mutant enzyme. An increased K(iNADPH) was measured for all of the mutant enzymes. The primary kinetic 13C-isotope effect is increased, while the primary deuterium kinetic isotope effect is decreased, indicating that the decarboxylation step has become more rate limiting under conditions where substrate is limiting. A quantitative analysis of the data suggests that the S128, H186, and N187 triad is multifunctional in the 6PGDH reaction and contributes as follows. The triad (1) participates in the precatalytic conformational change; (2) provides ground state binding affinity for 6PG and NADPH; and (3) affects the relative rates of reduction or decarboxylation of the 3-keto-6PG intermediate by anchoring the cofactor after hydride transfer, which is accompanied by the rotation of the nicotinamide ring around the N-glycosidic bond and displacement of C1 of 6PG, facilitating decarboxylation.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

17042485

Citation

Li, Lei, et al. "Role of the S128, H186, and N187 Triad in Substrate Binding and Decarboxylation in the Sheep Liver 6-phosphogluconate Dehydrogenase Reaction." Biochemistry, vol. 45, no. 42, 2006, pp. 12680-6.
Li L, Zhang L, Cook PF. Role of the S128, H186, and N187 triad in substrate binding and decarboxylation in the sheep liver 6-phosphogluconate dehydrogenase reaction. Biochemistry. 2006;45(42):12680-6.
Li, L., Zhang, L., & Cook, P. F. (2006). Role of the S128, H186, and N187 triad in substrate binding and decarboxylation in the sheep liver 6-phosphogluconate dehydrogenase reaction. Biochemistry, 45(42), 12680-6.
Li L, Zhang L, Cook PF. Role of the S128, H186, and N187 Triad in Substrate Binding and Decarboxylation in the Sheep Liver 6-phosphogluconate Dehydrogenase Reaction. Biochemistry. 2006 Oct 24;45(42):12680-6. PubMed PMID: 17042485.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Role of the S128, H186, and N187 triad in substrate binding and decarboxylation in the sheep liver 6-phosphogluconate dehydrogenase reaction. AU - Li,Lei, AU - Zhang,Lei, AU - Cook,Paul F, PY - 2006/10/18/pubmed PY - 2006/12/9/medline PY - 2006/10/18/entrez SP - 12680 EP - 6 JF - Biochemistry JO - Biochemistry VL - 45 IS - 42 N2 - Crystal structures of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver indicate that S128 and N187 are within hydrogen-bonding distance of 6PG in the E:6PG binary complex and NADPH in the E:NADPH binary complex. In addition, H186 is also within hydrogen-bonding distance of NADPH in the E:NADPH binary complex, while in the E:6PG binary complex it is within hydrogen-bonding distance of S128 and close to N187. The structures suggest that this triad of residues may play a dual role during the catalytic reaction. Site-directed mutagenesis has been performed to mutate each of the three residues to alanine. All mutant enzymes exhibit a decrease in V/E(t) (the turnover number), ranging from 7- to 67-fold. An increase in the Km for 6PG (K(6PG)) was observed for S128A and H187A mutant enzymes, while for the H186A mutation, K(6PG) is decreased by a factor of 2. K(NADP) remains the same as the wild type enzyme for the S128A and H186A mutant enzyme, while it increases by 6-fold in the N187A mutant enzyme. An increased K(iNADPH) was measured for all of the mutant enzymes. The primary kinetic 13C-isotope effect is increased, while the primary deuterium kinetic isotope effect is decreased, indicating that the decarboxylation step has become more rate limiting under conditions where substrate is limiting. A quantitative analysis of the data suggests that the S128, H186, and N187 triad is multifunctional in the 6PGDH reaction and contributes as follows. The triad (1) participates in the precatalytic conformational change; (2) provides ground state binding affinity for 6PG and NADPH; and (3) affects the relative rates of reduction or decarboxylation of the 3-keto-6PG intermediate by anchoring the cofactor after hydride transfer, which is accompanied by the rotation of the nicotinamide ring around the N-glycosidic bond and displacement of C1 of 6PG, facilitating decarboxylation. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/17042485/Role_of_the_S128_H186_and_N187_triad_in_substrate_binding_and_decarboxylation_in_the_sheep_liver_6_phosphogluconate_dehydrogenase_reaction_ L2 - https://dx.doi.org/10.1021/bi0613675 DB - PRIME DP - Unbound Medicine ER -