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Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver.
Eur J Biochem. 1986 May 02; 156(3):555-67.EJ

Abstract

The steady-state kinetics of the oxidative decarboxylation of 6-phosphogluconate catalysed by 6-phosphogluconate dehydrogenase from sheep liver in triethanolamine and phosphate buffers (pH 7.0) have been reinvestigated. In triethanolamine buffer the enzyme is inhibited by high NADP+ concentrations in the presence of low fixed concentrations of 6-phosphogluconate. Data are consistent with an asymmetric sequential mechanism in which NADP+ and 6-phosphogluconate bind randomly and product release is ordered. The pathway through the enzyme--6-phosphogluconate complex appears to be preferred in triethanolamine buffer. Pre-steady-state studies of the oxidative decarboxylation reaction at pH 6.0-8.0 show that hydride transfer is greater than 900 s-1. After the fast formation of NADPH in amounts equivalent to about half of the enzyme-active-centre concentration, the rate of NADPH formation is equal to the steady-state rate. Two possible interpretations are considered. Rapid fluorescence measurements of the displacement of NADPH from its complex with the enzyme at pH 6.0 and 7.0 indicate that the dissociation of NADPH is fast (greater than 800 s-1) and cannot be the rate-limiting step in oxidative decarboxylation. Coenzyme binding studies at equilibrium have been extended to include the determination of the dissociation constants for the binary complexes of enzyme with NADPH and NADP+ at pH 6.0-8.0 and the dissociation constant for NADPH in the ternary enzyme--6-phosphogluconate--NADPH complex in triethanolamine buffer, pH 7.0.

Authors

No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

3699023

Citation

Topham, C M., et al. "Kinetic Studies of 6-phosphogluconate Dehydrogenase From Sheep Liver." European Journal of Biochemistry, vol. 156, no. 3, 1986, pp. 555-67.
Topham CM, Matthews B, Dalziel K. Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver. Eur J Biochem. 1986;156(3):555-67.
Topham, C. M., Matthews, B., & Dalziel, K. (1986). Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver. European Journal of Biochemistry, 156(3), 555-67.
Topham CM, Matthews B, Dalziel K. Kinetic Studies of 6-phosphogluconate Dehydrogenase From Sheep Liver. Eur J Biochem. 1986 May 2;156(3):555-67. PubMed PMID: 3699023.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver. AU - Topham,C M, AU - Matthews,B, AU - Dalziel,K, PY - 1986/5/2/pubmed PY - 1986/5/2/medline PY - 1986/5/2/entrez SP - 555 EP - 67 JF - European journal of biochemistry JO - Eur. J. Biochem. VL - 156 IS - 3 N2 - The steady-state kinetics of the oxidative decarboxylation of 6-phosphogluconate catalysed by 6-phosphogluconate dehydrogenase from sheep liver in triethanolamine and phosphate buffers (pH 7.0) have been reinvestigated. In triethanolamine buffer the enzyme is inhibited by high NADP+ concentrations in the presence of low fixed concentrations of 6-phosphogluconate. Data are consistent with an asymmetric sequential mechanism in which NADP+ and 6-phosphogluconate bind randomly and product release is ordered. The pathway through the enzyme--6-phosphogluconate complex appears to be preferred in triethanolamine buffer. Pre-steady-state studies of the oxidative decarboxylation reaction at pH 6.0-8.0 show that hydride transfer is greater than 900 s-1. After the fast formation of NADPH in amounts equivalent to about half of the enzyme-active-centre concentration, the rate of NADPH formation is equal to the steady-state rate. Two possible interpretations are considered. Rapid fluorescence measurements of the displacement of NADPH from its complex with the enzyme at pH 6.0 and 7.0 indicate that the dissociation of NADPH is fast (greater than 800 s-1) and cannot be the rate-limiting step in oxidative decarboxylation. Coenzyme binding studies at equilibrium have been extended to include the determination of the dissociation constants for the binary complexes of enzyme with NADPH and NADP+ at pH 6.0-8.0 and the dissociation constant for NADPH in the ternary enzyme--6-phosphogluconate--NADPH complex in triethanolamine buffer, pH 7.0. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/3699023/Kinetic_studies_of_6_phosphogluconate_dehydrogenase_from_sheep_liver_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0014-2956&date=1986&volume=156&issue=3&spage=555 DB - PRIME DP - Unbound Medicine ER -