Elementary steps in the reaction mechanism of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: kinetics of acetylation and deacetylation.

Published

Journal Article

The kinetics of the acetylation of the pyruvate dehydrogenase complex from Escherichia coli by [3-14C]pyruvate and of the deacetylation of the complex by coenzyme A have been studied by using rapid mixing-quench techniques. The time course for acetylation in 4 mM thiamin pyrophosphate, 2 mM MgSO4, and 0.02 M potassium phosphate (pH 7.0) at 4 degrees C can be analyzed in terms of two kinetic processes. At long times 10 nmol of acetyl groups is incorporated per mg of enzyme complex (48 sites per complex of molecular weight 4.8 X 10(6)). The slower process is much too slow to be of catalytic significance. The rate constant for the faster process is not dependent on enzyme concentration and reaches a limiting value of 40--65 s-1 at high pyruvate concentrations; the exact value is dependent on the detailed acetylation mechanism assumed. The minimum molar turnover number of the enzyme complex is 420 s-1 (17.5 s-1 per pyruvate decarboxylase). The acetylated lipoic acids are deacetylated by coenzyme A at a rate much faster than that of acetylation. Complete deacetylation is obtained only if the deacetylation is carried out within seconds of the acetylation, apparently because dead-end intramolecular transfers of acetyl groups from the lipoic acids to other functional groups on the enzyme not essential for catalytic activity can occur. The results obtained suggest only about half of the acetylation reactions are on the main catalytic pathway.

Full Text

Duke Authors

Cited Authors

  • Akiyama, SK; Hammes, GG

Published Date

  • September 2, 1980

Published In

Volume / Issue

  • 19 / 18

Start / End Page

  • 4208 - 4213

PubMed ID

  • 6998493

Pubmed Central ID

  • 6998493

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00559a011

Language

  • eng

Conference Location

  • United States