title: Influence of multiple cysteines on human 3-hydroxy-3-methylglutaryl-CoA lyase activity and formation of inter-subunit adducts.
abstract: Human 3-hydroxy-3-methylglutaryl-CoA lyase catalyzes formation of acetyl-CoA and acetoacetate in a reaction that requires divalent cation and is stimulated by sulfhydryl protective reagents. The enzyme is a homodimer and inter-subunit adducts form in the absence of reducing agents or upon treatment with cysteine selective crosslinking agents. To address the influence of cysteines on enzyme activity and formation of inter-subunit and intra-subunit adducts, single serine substitutions have been engineered for each enzyme cysteine. Enzyme activity varies for each cysteine→serine mutant protein and different mutations have widely different effects on recovery of activity upon DTT treatment of non-reduced enzyme. These levels of enzyme activity do not strongly correlate with formation of inter-subunit adducts by these HMGCL mutants. C170S, C266S, and C323S proteins do not form inter-subunit disulfide adducts but such an adduct is restored in the C170S/C174S double mutant. Coexpression of HMGCL proteins encoded by C266S and C323S expression plasmids supports formation of a C266S/C323S heterodimer which does form a covalent inter-subunit adduct. These observations are interpreted in the context of competition between cysteines in formation of intra-subunit and inter-subunit heterodisulfide adducts.
authors.affiliation: Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City, MO 64110, United States.
authors.forename: Christa
authors.initials: C
authors.lastname: Montgomery
authors.forename: Henry M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D003545:Cysteine