title: 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.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
authors.forename: P W
authors.initials: PW
authors.lastname: Hruz
authors.forename: H M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D000214:Acyl Coenzyme A / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001717:Birds; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
chemical_list: D000214:Acyl Coenzyme A; D004220:Disulfides; D007461:Iodoacetates; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C075847:2-butynoyl-coenzyme A; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D003545:Cysteine; D019807:Iodoacetic Acid