General acid/base catalysis by a histidine residue of mammalian and bacterial HMG-CoA reductase
Abstract
By investigating the pH-variation of kinetic parameters, Veloso, Cleland, and Porter (Biochemistry 20, 887-894, 1981) postulated that a histidine residue participates in catalysis by yeast 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Histidine modifying reagents also inactivate yeast HMG-CoA reductase (Dugan and Katiyar, Biochem. Biophys. Res. Commun. 141, 278-284, 1986). I therefore used chemical modification and site-directed mutagenesis to identify a catalytic site histidine of HMG-CoA reductase of Pseudomonas mevalonii (Ps. HMGR) and of the catalytic domain of the Syrian hamster enzyme (R$\sb{\rm cat}$). Ps. HMGR and R$\sb{\rm cat}$ were overexpressed in Escherichia coli, purified to homogeneity, and characterized. Diethyl pyrocarbonate (DEPC) inactivated both enzymes, and hydroxylamine partially restored activity. Sequence comparisons revealed that only His$\sp{381}$ (Ps. HMGR) and His$\sp{865}$ (R$\sb{\rm cat}$) are totally conserved among the catalytic domains of all known HMG-CoA reductases. The codon for His$\sp{381}$ was changed to the codons for alanine, lysine, asparagine, and glutamine, and that for His$\sp{865}$ to the codons for lysine and glutamine. Following overexpression in E. coli, all mutant enzymes were purified to homogeneity. While all mutant enzymes exhibited less than 1.5% of wild-type catalytic activity, all chromatographed on substrate affinity supports like wild-type enzyme, and K$\sb{\rm m}$ values approximated those for wild-type enzyme. In addition, Ps. HMGR mutant enzymes exhibited wild-type crystal morphology. I therefore infer that the low catalytic activity of these mutant enzyme was not a result of gross conformational changes. Whereas His$\sp{381}$ mutant enzymes were not inactivated by DEPC, His$\sp{865}$ mutant enzymes were sensitive to DEPC. pK$\sb1$ values were determined for all Ps. HMGR mutant enzymes. Mutant enzyme Ps. HMGR H381K exhibited an elevated pK$\sb1$ of 10.2, consistent with lysine acting as a general base at high pH. Exogenous amines enhanced the activity of Ps. HMGR H381A in a pH-dependent manner, suggesting that the unprotonated amine acts as the general base in catalysis. His$\sp{381}$ of Ps. HMGR and His$\sp{865}$ of R$\sb{\rm cat}$, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xxx-Xxx-Asn-Arg-Ser motif of the catalytic domain of all eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.
Degree
Ph.D.
Advisors
Rodwell, Purdue University.
Subject Area
Biochemistry|Molecular biology|Biology
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