THE ACYL CARRIER PROTEIN OF MALONATE DECARBOXYLASE OF MALONOMONAS-RUBRA CONTAINS 2'-(5''-PHOSPHORIBOSYL)-3'-DEPHOSPHOCOENZYME-A AS A PROSTHETIC GROUP

Malonate decarboxylase of Malonomonas rubra is composed of soluble and membrane-bound components and contains an acetyl residue that is esse ntial for catalytic activity, Upon incubation with hydroxylamine, the acetyl residue is removed, forming an inactive thiol enzyme, which is reactivated by acetylation with ATP, acetate, and a specific ligase, A fter incubation of the thiol enzyme with iodoacetate in the presence o f excess dithioerythritol, the prosthetic group thiol residue was carb oxymethylated and reactivation by acetylation was impaired. Radioactiv e labeling with [1-C-14]iodoacetate revealed the site of carboxymethyl ation on a distinct cytoplasmic protein with the apparent molecular ma ss of 14 000 Da, The same protein was specifically labeled by enzymic acetylation of the thiol enzyme with [1-C-14]acetate and ATP. Malonate decarboxylation by [C-14]acetyl malonate decarboxylase resulted in th e release of the radioactive acetyl residue from the enzyme, indicatin g that this acetyl residue is exchanged for a malonyl residue during c atalysis, The acyl carrier protein has been purified as its [C-14]carb oxymethylated derivative to apparent homogeneity. The prosthetic group of the acyl carrier protein was isolated after alkaline hydrolysis, a nd its chemical structure was identified by high-performance liquid ch romatography (HPLC) with the corresponding compound from citrate lyase from Klebsiella pneumoniae as reference and by mass spectrometry. Mal onate decarboxylase was found to carry the same prosthetic group as ci trate lyase, i.e. 2'-(5double prime-phosphoribosyl)-3'-dephospho-CoA.