J. Biol. Chem., Vol. 260, Issue 27, 14610-14615, 11, 1985
Pyruvate dehydrogenase complex of Escherichia coli. Thiamin pyrophosphate and NADH-dependent hydrolysis of acetyl-CoA
CA CaJacob, GR Gavino and PA Frey
When the pyruvate dehydrogenase complex of Escherichia coli is reduced by
NADH and alkylated by N-[14C]ethylmaleimide, 19-20 nmol of N-
[14C]ethylmaleimide are bound per mg of complex. This is in accord with the
presence of 10 nmol of functional lipoyl moieties per mg of complex as
previously reported. Thus the lipoyl groups are all coupled via
dihydrolipoyl dehydrogenase (E3) to reduction by NADH. As previously
reported, the complex reductively acetylated by pyruvate and containing 10
nmol of acetyldihydrolipoyl groups per mg of complex produces about 5 nmol
of NADH/mg of complex when challenged with CoA and NAD+ in a fast burst.
Under anaerobic conditions a slow secondary process extending over 1 h
produces another 5 nmol of NADH/mg of complex. The relationship between the
two classes of acetyldihydrolipoyl groups is unknown but could reflect
either intrinsic structural inequivalence of lipoyl groups (2/subunit of
dihydrolipoyl transacetylase, E2). Alternatively, the acetyldihydrolipoyl
groups may undergo reversible isomerization to structurally distinct forms.
The purified complex catalyzes the cleavage of acetyl-CoA by two processes.
The trace contaminant phosphotransacetylase catalyzes cleavage by phosphate
to acetyl-P. The complex itself catalyzes hydrolysis of acetyl-CoA in a
reaction that requires all three enzymes, NADH, thiamin pyrophosphate, and
the lipoyl groups of E2. The hydrolytic pathway evidently involves overall
reversal of the reaction, leading ultimately to the formation of
acetyl-thiamin pyrophosphate, which undergoes hydrolysis to acetate.
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