Abstract
A procedure is described for the purification of phosphopyruvate (
PEP
) carboxylase (EC 4.1.1.31) from extracts of
Escherichia coli
, strain W. The enzyme, purified 160-fold, catalysed the stoicheiometric formation of oxaloacetate from phosphopyruvate and potassium bicarbonate. This reaction occurred most readily at pH 8·5, and required the presence of divalent metal ions (Mg
2+
≥ Mn
2+
> Co
2+
); the
K
m
for Mg
2+
was 9·8 x 10
-4
M. Addition of acetylcoenzyme
A
to the reaction system greatly stimulated the rate of oxaloacetate formation. At saturating concentrations of acetyl-coenzyme
A
(about 1 mM), the reaction rate was thirty times more rapid than that observed if the thiol-ester was omitted; other acyl-coenzyme
A
derivatives were less effective in stimulating
PEP
-carboxylase activity (acetyl- > propionyl- > butyryl- ≫ acrylyl- > crotonyl-coenzyme
A
). The
K
m
for acetyl-coenzyme
A
was 1·4 x 10
-4
M. The effect of acetyl-coenzyme
A
was catalytic, and increased the apparent affinity of the enzyme for
PEP
from
K
m
= 5.5 x 10
-3
M to
K
m
= 6·4 x 10
-4
M. Since 1 M-urea inhibited the acetyl-coenzyme
A
-stimulated carboxylation of
PEP
but did not affect the enzymic activity in the absence of acetyl-coenzyme
A
, it is suggested that acetyl-coenzyme
A
exerts its stimulatory effect through interaction with an allosteric site on the enzyme. The results obtained also suggest a mechanism for the physiological regulation of
PEP
-carboxylase activity in
E. coli
.
Cited by
79 articles.
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