Avian 3-hydroxy-3-methylglutaryl-CoA lyase: sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines.

ABSTRACT

Catalysis by purified avian 3-hydroxy-3-methylglutaryl-CoA lyase is critically dependent on the reduction state of the enzyme, with less than 1% of optimal activity being observed with the air-oxidized enzyme. The enzyme is irreversibly inactivated by sulfhydryl-directed reagents with the rate of this inactivation being highly dependent upon the redox state of a critical cysteine. Methylation of reduced avian lyase with 1 mM 4-methylnitrobenzene sulfonate results in rapid inactivation of the enzyme with a k(inact) of 0.178 min-1. The oxidized enzyme is inactivated at a sixfold slower rate (k(inact) = 0.028 min-1). Inactivation of the enzyme with the reactive substrate analog 2-butynoyl-CoA shows a similar dependence upon the enzyme's redox state, with a sevenfold difference in k(inact) observed with oxidized vs. reduced forms of the enzyme. Chemical cross-linking of the reduced enzyme with stoichiometric amounts of the bifunctional reagents 1,3-dibromo-2-propanone (DBP) or N,N'-ortho-phenylene-dimaleimide (PDM) coincides with rapid inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme treated with bifunctional reagent reveals a band of twice the molecular weight of the lyase monomer, indicating that an intersubunit cross-link has been formed. Differential labeling of native and cross-linked protein with [1-14C]iodoacetate has identified as the primary cross-linking target a cysteine within the sequence VSQAACR, which maps at the carboxy-terminus of the cDNA-deduced sequence of the avian enzyme (Mitchell, G.A., et al., 1991, Am. J. Hum. Genet. 49, 101). In contrast, bacterial HMG-CoA lyase, which contains no corresponding cysteine, is not cross-linked by comparable treatment with bifunctional reagent. These results provide evidence for a potential regulatory mechanism for the eukaryotic enzyme via thiol/disulfide exchange and identify a cysteinyl residue with the reactivity and juxtaposition required for participation in disulfide formation.