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()

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.