M. Berg et al., THE ACYL CARRIER PROTEIN OF MALONATE DECARBOXYLASE OF MALONOMONAS-RUBRA CONTAINS 2'-(5''-PHOSPHORIBOSYL)-3'-DEPHOSPHOCOENZYME-A AS A PROSTHETIC GROUP, Biochemistry, 35(15), 1996, pp. 4689-4696
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.