Mechanism of Reconstitution of Brewers' Yeast Pyruvate Decarboxylase with Thiamin Diphosphate and Magnesium

Joseph A. Vaccaro, Edward J. Crane, Thomas K. Harris, Michael W. Washabaugh

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and α4 isozyme (α4-PDC) exhibits biphasic kinetics with 52 ± 11% of the PDC reacting with k1 = (1.0 ± 0.3) × 10-2 s-1 and 48 ± 12% of the PDC reacting with k2 = (1.1 ± 0.6) × 10-1 s-1 when TDP (KTDP = 0.5 ± 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 ± 0.5) × 10-2 s-1 upon addition of Mg2+ (KMg2+ = 0.2 ± 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 ≤3-fold and has no effect on the value of k1. These results are summarized in a model for apo-PDC reconstitution, and implications for mechanistic studies on PDC are discussed.

Original languageEnglish (US)
Pages (from-to)12636-12644
Number of pages9
JournalBiochemistry
Volume34
Issue number39
DOIs
StatePublished - Oct 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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