3-HYDROXY-3-METHYLGLUTARYL-COA LYASE - EXPRESSION AND ISOLATION OF THE RECOMBINANT HUMAN ENZYME AND INVESTIGATION OF A MECHANISM FOR REGULATION OF ENZYME-ACTIVITY

cDNA encoding the mature form of human hydroxymethylglutaryl-CoA (HMG- CoA) lyase, a mitochondrial matrix protein, has been used to prepare e xpression plasmids appropriate for production of this protein in Esche richia coli. Using a T7 RNA polymerase-based pET system, HMG-CoA lyase was overexpressed but largely recovered in an insoluble, catalyticall y inactive form. In contrast, an expression plasmid (pTrcHL-1), derive d from pTrc99a, supported production of soluble, active enzyme. A synt hetic oligonucleotide cassette was employed to produce an enzyme varia nt in which cysteine was replaced by serine at position 323. Both wild -type and C323S HMG-CoA lyases were isolated in homogeneous form and c haracterized. The function of Cys-323 in influencing catalytic activit y in vitro has been investigated by comparing the response of wild-typ e and C323S lyases to oxidation and reduction. Additionally, the con s equences of treatment of these enzymes with the sulfhydryl-directed bi functional reagent, o-phenylenedimaleimide have been determined. The r esults support the hypothesis that a thiol/disulfide exchange mechanis m affects enzyme activity in vitro and indicate that Cys-323 residues on adjacent subunits of the homodimeric native enzyme are suitably pos itioned to form an intersubunit cross-link upon oxidative inactivation and disulfide formation.