title: Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase.
abstract: Thimet oligopeptidase is a metalloenzyme involved in regulating neuropeptide processing. Three cysteine residues (246, 248, 253) are known to be involved in thiol activation of the enzyme. In contrast to the wild-type enzyme, the triple mutant (C246S/C248S/C253S) displays increased activity in the absence of dithiothreitol. Dimers, purportedly formed through cysteines 246, 248 and 253, have been thought to be inactive. However, analysis of the triple mutant by native gel electrophoresis reveals the existence of dimers and multimers, implying that oligomer formation is mediated by other cysteines, probably on the surface, and that some of these forms are enzymatically active. Isolation and characterization of iodoacetate-modified monomers and dimers of the triple mutant revealed that, indeed, certain dimeric forms of the enzyme are still fully active, whereas others show reduced activity. Cysteine residues potentially involved in dimerization were identified by modeling of thimet oliogopeptidase to its homolog, neurolysin. Five mutants were constructed; all contained the triple mutation C246S/C248S/C253S and additional substitutions. Substitutions at C46 or C682 and C687 prevented multimer formation and inhibited dimer formation. The C46S mutant had enzymatic activity comparable to the parent triple mutant, whereas that of C682S/C687S was reduced. Thus, the location of intermolecular disulfide bonds, rather than their existence per se, is relevant to activity. Dimerization close to the N-terminus is detrimental to activity, whereas dimerization near the C-terminus has little effect. Altering disulfide bond formation is a potential regulatory factor in the cell owing to the varying oxidation states in subcellular compartments and the different compartmental locations and functions of the enzyme.
authors.affiliation: Department of Chemistry, Wellesley College, Wellesley, MA 02481, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Sigman
authors.forename: M L
authors.initials: ML
authors.lastname: Sharky
authors.forename: S T
authors.initials: ST
authors.lastname: Walsh
authors.forename: A
authors.initials: A
authors.lastname: Pabon
authors.forename: M J
authors.initials: MJ
authors.lastname: Glucksman
authors.forename: A J
authors.initials: AJ
authors.lastname: Wolfson
mesh_terms: D019943:Amino Acid Substitution; D003545:Cysteine; D019281:Dimerization; D004789:Enzyme Activation; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D040681:Structural Homology, Protein
chemical_list: D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; C062316:neurolysin; D003545:Cysteine