PURIFICATION AND CHARACTERIZATION OF A CYTOPLASMIC ENZYME COMPONENT OF THE NA-ACTIVATED MALONATE DECARBOXYLASE SYSTEM OF MALONOMONAS-RUBRA,ACETYL-S-ACYL CARRIER PROTEIN, MALONATE ACYL CARRIER PROTEIN-SH TRANSFERASE()
H. Hilbi et P. Dimroth, PURIFICATION AND CHARACTERIZATION OF A CYTOPLASMIC ENZYME COMPONENT OF THE NA-ACTIVATED MALONATE DECARBOXYLASE SYSTEM OF MALONOMONAS-RUBRA,ACETYL-S-ACYL CARRIER PROTEIN, MALONATE ACYL CARRIER PROTEIN-SH TRANSFERASE(), Archives of microbiology, 162(1-2), 1994, pp. 48-56
Malonate decarboxylation by crude extracts of Malonomonas rubra was sp
ecifically activated by Na+ and less efficiently by Li+ ions. The extr
acts contained an enzyme catalyzing CoA transfer from malonyl-CoA to a
cetate, yielding acetyl-CoA and malonate. After about a 26-fold purifi
cation of the malonyl-CoA:acetate CoA transferase, an almost pure enzy
me was obtained, indicating that about 4% of the cellular protein cons
isted of the CoA transferase. This abundance of the transferase is in
accord with its proposed role as an enzyme component of the malonate d
ecarboxylase system, the key enzyme of energy metabolism in this organ
ism. The apparent molecular weight of the polypeptide was 67,000 as re
vealed from SDS-polyacrylamide gel electrophoresis. A similar molecula
r weight was estimated for the native transferase by gel chromatograph
y, indicating that the enzyme exists as a monomer. Kinetic analyses of
the CoA transferase yielded the following: pH-optimum at pH5.5, an ap
parent K-m for malonyl-CoA of 1.9 mM, for acetate of 54 mM, for acetyl
-CoA of 6.9 mM, and for malonate of 0.5 mM. Malonate or citrate inhibi
ted the enzyme with an apparent K-i of 0.4 mM and 3.0 mM, respectively
. The isolated CoA transferase increased the activity of malonate deca
rboxylase of a crude enzyme system, in which part of the endogenous Co
A transferase was inactivated by borohydride, about three-fold. These
results indicate that the CoA transferase functions physiologically as
a component of the malonate decarboxylase system, in which it catalyz
es the transfer of acyl carrier protein from acetyl acyl carrier prote
in and malonate to yield malonyl acyl carrier protein and acetate. Mal
onate is thus activated on the enzyme by exchange for the catalyticall
y important enzyme-bound acetyl thioester residues noted previously. T
his type of substrate activation resembles the catalytic mechanism of
citrate lyase and citramalate lyase.