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

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‐phenylenedimaleimide (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.