title: 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.
abstract: beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but 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 [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins 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.
authors.affiliation: Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark.
authors.forename: K A
authors.initials: KA
authors.lastname: McGuire
authors.forename: M
authors.initials: M
authors.lastname: Siggaard-Andersen
authors.forename: M G
authors.initials: MG
authors.lastname: Bangera
authors.forename: J G
authors.initials: JG
authors.lastname: Olsen
authors.forename: P
authors.initials: P
authors.lastname: von Wettstein-Knowles
mesh_terms: D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002845:Chromatography; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019814:Myristic Acid / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D019814:Myristic Acid; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D004247:DNA; C078182:beta-ketoacyl-acyl carrier protein synthase I; D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase; D008239:Lysine; D003545:Cysteine