HIS(865) IS THE CATALYTICALLY IMPORTANT HISTIDYL RESIDUE OF SYRIAN-HAMSTER 3-HYDROXY-3-METHYLGLUTARYL-COENZYME-A REDUCTASE

Involvement in catalysis of a histidyl residue of Syrian hamster 3-hyd roxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was suggested by the ability of diethyl pyrocarbonate to abolish catalytic activity, ac companying spectral changes, and reactivation by hydroxylamine. The 7 histidines present in the catalytic domain of the hamster enzyme were changed to glutamine (His474, His487, His634, His751, His860, and His8 65), lysine (His865), or tyrosine (His868). Overexpression in Escheric hia coli yielded six soluble mutant proteins, one insoluble protein (H 634Q), and one which was degraded in vivo (H487Q). Following purificat ion to homogeneity, mutant enzymes H474Q, H751Q, H860Q, and H868Y had essentially wild-type catalytic activity, while mutant enzymes H865K a nd H865Q had less than 0.6% wild-type activity. The low activity of mu tant enzymes H865K and H865Q is unlikely to reflect altered structural integrity since both chromatographed on affinity supports like wild-t ype enzyme and had K(m) values for (S)-HMG-CoA (31 and 16 muM) and for NADPH (60 and 24 muM) close to those for wild-type enzyme (31 and 52 muM for (S)-HMG-CoA and NADPH, respectively). His865 of hamster HMG-Co A reductase, the histidine of the consensus Leu-Val-Xaa-Ser-His-Met-Xa a-Xaa-Asn-Arg-Ser motif and the only histidine conserved among the cat alytic domains of all HMG-CoA reductases, thus appears to be a general acid/base functional in catalysis.