Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either.
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An et al. (1999) studied this question.