Pyruvate Formate-Lyase of Escherichia coli: the Acetyl-Enzyme Intermediate

Authors

  • Joachim KNAPPE,

    1. Institut für Biologische Chemie der Universität Heidelberg, D-6900 Heidelberg 1, Im Neuenheimer Feld 501, Federal Republic of Germany
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  • Hans P. BLASCHKOWSKI,

    1. Institut für Biologische Chemie der Universität Heidelberg, D-6900 Heidelberg 1, Im Neuenheimer Feld 501, Federal Republic of Germany
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  • Peter GRÖBNER,

    1. Institut für Biochemie und Experimentelle Krebsforschung der Universität Innsbruck, A-6020 Innsbruck, Peter-Mayer-Straße 2, Austria
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  • Thomas SCHMTTT

    1. Institut für Biologische Chemie der Universität Heidelberg, D-6900 Heidelberg 1, Im Neuenheimer Feld 501, Federal Republic of Germany
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Abstract

The reaction pyruvate + CoA ⇌ acetyl-CoA + formate, catalyzed by pyruvate formate-lyase of Escherichia coli, occurs by the succeeding half-reactions (a) E + pyruvate ⇌ E-acetyl + formate; (b) E-acetyl + CoA ⇌ E + acetyl-CoA.

Making use of coupled optical assays, a ‘ping-pong mechanism’ was derived from the complete kinetic investigation of the forward and reverse reactions. The thermodynamic equilibrium constant of the overall reaction was calculated from the kinetic constants to be K= 750 (30°C, pH 8.1), which agrees with chemically determined values.

The intermediate acetyl-enzyme, which had been previously proposed from the [14C]formate-pyruvate exchange, was detected by product-pulse experiments with [2-14C]pyruvate and trapped by acid precipitation. The acetyl group is linked to a sulfhydryl group of the protein.

The value of the equilibrium constant of the first half-reaction is about 50, as directly measured and calculated from the kinetic data. It was concluded that the standard free energy of hydrolysis of acetyl-enzyme is about 1.7 kcal (7.1 kJ) more negative than that of acetyl-CoA.

The intermediate was found to react with dithiothreitol with a second-order rate constant at 30°C and pH 7.6 of 1160 M−1× min−1. It resulted in a half-life of 4 s (or 20 s at 0°C) in the particular buffer which was required for enzyme stabilization.

The enzyme (about 60 U/mg) was prepared by carrying its purified inactive form through the enzyme-II-dependent activation reaction, employing photoreduced flavodoxin along with the effector compounds S-adenosylmethionine and oxamate (as a pyruvate analogue).

Abbreviations
Mops

2-(N-morpholino)propanesulfonate

Enzymes
 

(CBN recommendations 1972). Pyruvate formate-lyase (CoA-acetylating) (EC 4.1.–.– or 2.3.1.–)

 

pyruvate dehydrogenase complex = pyruvate dehydrogenase (EC 1.2.4.1) plus dihydrolipoamide transacetylase (EC 2.3.1.12) plus dihydrolipoamide dehydrogenase (EC 1.6.4.3)

 

pyruvate :flavodoxin oxidoreductase (CoA-acetylating) (EC 1.2.7.–)

 

lactate dehydrogenase (EC 1.1.1.27)

 

malate dehydrogenase (EC 1.1.1.37)

 

phosphate acetyltransferase (EC 2.3.1.8)

 

citrate synthase (EC 4.1.3.7)

Ancillary