title: A structural limitation on enzyme activity: the case of HMG-CoA synthase.
abstract: Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine. Subsequent enzymatic studies showed that the overall reaction rate of the enzyme was increased 140-fold. An X-ray crystallographic study of the Ala110Gly mutant enzyme demonstrated unanticipated adjustments in the active site that resulted in additional stabilization of all three steps of the reaction pathway. The rates of acetylation and hydrolysis of the mutant enzyme increased because the amide nitrogen of Ser308 shifts 0.4 A toward the catalytic cysteine residue. This motion positions the nitrogen to better stabilize the intermediate negative charge that develops on the carbonyl oxygen of the acetyl group during both the formation of the acyl-enzyme intermediate and its hydrolysis. In addition, the hydroxyl of Ser308 rotates 120 degrees to a position where it is able to stabilize the carbanion intermediate formed by the methyl group of the acetyl-S-enzyme during its condensation with acetoacetyl-CoA.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA. csteussy@purdue.edu
authors.forename: Calvin N
authors.initials: CN
authors.lastname: Steussy
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Robison
authors.forename: Alison M
authors.initials: AM
authors.lastname: Tetrick
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Knight
authors.forename: Victor W
authors.initials: VW
authors.lastname: Rodwell
authors.forename: Cynthia V
authors.initials: CV
authors.lastname: Stauffacher
authors.forename: Autumn L
authors.initials: AL
authors.lastname: Sutherlin
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D018360:Crystallography, X-Ray; D019281:Dimerization; D013293:Enterococcus faecalis / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D000105:Acetyl Coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase