3-Hydroxy-3-methylglutaryl-CoA synthase. A role for glutamate 95 in general acid/base catalysis of C-C bond formation.
Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acyl-CoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by (13)C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu(95) functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK(a) of Glu(95) as required to support its function as a general acid.
Chun
K Y
KY
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Vinarov
D A
DA
Zajicek
J
J
Miziorko
H M
HM
D000214:Acyl Coenzyme A / Q000378:metabolism; D000478:Alkylation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D018698:Glutamic Acid / Q000378:metabolism*; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D000214:Acyl Coenzyme A; D018698:Glutamic Acid; C046467:3-chloropropionyl-coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase