3-hydroxy-3-methylglutaryl-CoA synthase: Participation of invariant acidicresidues in formation of the acetyl-S-enzyme reaction intermediate

Inactivation of HMG-CoA synthase by a carboxyl-directed reagent, 1-ethyl-3- (3-dimethyl-aminopropyl)carbodiimide (EDC), in a concentration-dependent an d substrate-protectable manner suggested that the active site contains reac tive acidic amino acids. This observation prompted functional evaluation of 11 invariant acidic amino acids by site-directed mutagenesis. Characteriza tion of the isolated synthase variants' ability to catalyze overall and par tial reactions identified three mutant synthases (D99A, D159A, and D203A) t hat exhibit significant diminution of k(cat) for the overall reaction (10(2 )-, 10(3)-, and 10(4)-fold decreases, respectively). D99A, D159A, and D203A form the acetyl-S-enzyme intermediate very slowly (0.0025, 0.0026, 0.0015 U/mg, respectively, measured at pH 7.0 and 22 degreesC) as compared to the wild-type synthase (1.59 U/mg), where intermediate formation approaches rat e-limiting status. Differences in substrate saturation do not account for i mpaired activities or rates of intermediate formation. The structural integ rity of the purified mutants' active sites is demonstrated by their abiliti es to bind a spin-labeled acyl-CoA analogue (R.CoA) with affinities and sto ichiometries comparable to values measured for wild-type synthase. The impa ct of three distinct amino acids on reaction intermediate formation support s a mechanism of acetyl-S-enzyme formation that probably requires formation and directed collapse of a tetrahedral adduct. O-18-induced shift of the C -13 NMR signal of C-13 acetyl-S-enzyme demonstrates that an analogous tetra hedral species is produced upon solvent exchange with the acetyl-S-enzyme. Partial discrimination between the functions of D99, D159, and D203 becomes possible based on the observation that D159A and D203A synthases exhibit r etarded kinetics of solvent O-18 exchange while D99A fails to support O-18 exchange.