beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: Aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket

beta -Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbo n bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the a cyl primer substrate. Six residues of Escherichia coli beta -ketoacyl-ACP s ynthase I (KAS I) implicated in these reactions were subjected to site-dire cted mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the follow ing conclusions. The active site Cys-163 is not required for decarboxylatio n, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable trans fer of the acyl primer substrate. Maintenance of the structural integrity o f the active site by Asp-306 and Glu-309 is required for decarboxylation bu t not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Ol sen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis re vealed by the mutant analyses is that the acyl primer transfer activity of beta -ketoacyl-ACP synthase I is inhibited by free ACP at physiological con centrations. Differences in the inhibitory response by individual mutant pr oteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309 , Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.