Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme a reductase

We identify His³⁸¹ of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His³⁸¹ of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His³⁸¹ to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His³⁸¹ mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (V_max 0.06-0.5% that of wild-type enzyme), but K_m values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, K_m values at pH 8.1 were 0.45, 0.27,3.7, and 0.71 mM [(-R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD⁺]. K_m values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD⁺]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK₁ and by 0.5-1.3 pH units for pK₂). For mutant enzyme H381K, the pK₁ of 10.2 is consistent with lysine acting as a general base at high pH. His³⁸¹ of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.