Identification, Localization, and Function of the Thiamin Pyrophosphate and Flavin Adenine Dinucleotide Dependent Pyruvate Oxidase in Isolated Membrane Vesicles of Escherichia coli B†

Lauraine A. Shaw-Goldstein, Robert B. Gennis, Christopher Walsh

Research output: Contribution to journalArticle

Abstract

The enzymatic activity responsible for pyruvate-dependent O2 uptake in membrane vesicles of E. coli B is identified as the thiamin pyrophosphate requiring flavoprotein pyruvate oxidase, a peripheral membrane enzyme which oxidatively decarboxylates pyruvate to acetate and CO2. Enzyme activity was inhibited by antibody to authentic pyruvate oxidase (from E. coli W191-6) after solubilization with Triton X-100, but was insensitive to antibody added exogenously either to freshly prepared isolated cytoplasmic membrane vesicles or to spheroplasts. This latency suggests an essentially exclusive location of the membrane-associated pyruvate oxidase at the inner face of the cytoplasmic membranes. This membrane location was confirmed using analogues of thiamin pyrophosphate, thiamin thiazalone pyrophosphate, and thiamin thiothiazolone pyrophosphate. These coenzyme analogues are membrane impermeant. While they show Ki values of 10-7 and 2 × 10-8 M for solubilized E. coli B pyruvate oxidase, they cause no more than 20% inhibition of vesicle-associated enzyme at high exogenous concentrations. The metabolic function of pyruvate oxidase is not the provision of activated acetyl groups (the role of the pyruvate dehydrogenase multienzyme complex) but appears rather to provide substrate-derived electrons to the membrane respiratory chain for the generation of the electrochemical potential, so that ΔμH+ can be used to drive solute active transport (shown here for amino acid transport). Exogenous addition of solubilized pyruvate oxidase to isolated membrane vesicles results in weak binding and apparent saturation, as judged by a 25-fold increase in pyruvate-stimulated O2 uptake. However, there is only a 1.6-fold stimulation in the rate of active transport of amino acids, indicating a much lower coupling efficiency of exogenous enzyme. For example, we calculate that for endogenous (cytoplasmic side) pyruvate oxidase some 55 electron pairs pass down the membrane electron-transport chain per molecule of proline actively transported; with exogenous pyruvate oxidase, 510 electron pairs are required per molecule of proline actively accumulated.

Original languageEnglish (US)
Pages (from-to)5605-5613
Number of pages9
JournalBiochemistry
Volume17
Issue number26
DOIs
StatePublished - Jan 1 1978
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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