title: Sulfhydryl-directed triggering of conformational changes in the enzyme rhodanese.
abstract: The enzyme rhodanese in the form without transferred sulfur, (E), was inactivated by carboxymethylation with iodoacetic acid (E.IAA), and its conformation was compared with that of E inactivated by oxidative processes (Eox). Formation of E.IAA led to the exposure of binding sites for the fluorescent apolar probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (BisANS). The dissociation constant for BisANS decreased as the concentration of E.IAA decreased and ranged from approximately 200 microM at 1 mg/ml protein to approximately 2 microM at protein concentrations below 0.1 mg/ml. Centrifugation confirmed that E.IAA, but not the underivatized enzyme, could associate. E.IAA was proteolyzable by chymotrypsin or endoproteinase Glu C (V8), while rhodanese containing bound sulfur, ES, was totally refractory, and E was only clipped to a small extent. This constellation of consequences was only previously observed with oxidatively inactivated rhodanese. Fluorescence depolarization measurements of bound BisANS were consistent with exposure of apolar surfaces and association of the protein. The fluorescence spectra of BisANS bound to E.IAA or Eox were identical and distinct from the spectrum of BisANS bound to phenylglyoxal-inactivated ES. Digestion with chymotrypsin was followed using protein and BisANS fluorescence and showed a similar response for E.IAA and Eox. These results indicate that the consequences of forming Eox and E.IAA are very similar. Thus, reaction of the active site sulfhydryl group apparently triggers a conformational change leading to increased protein flexibility and increased exposure of hydrophobic surfaces. In the case of oxidation, the trigger might involve initial formation of an active site sulfenic acid which ultimately gives higher oxidation states that could include disulfides.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
authors.forename: P M
authors.initials: PM
authors.lastname: Horowitz
authors.forename: N L
authors.initials: NL
authors.lastname: Criscimagna
mesh_terms: D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology*; D019807:Iodoacetic Acid; D007700:Kinetics; D010658:Phenylglyoxal / Q000494:pharmacology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013466:Sulfurtransferases / Q000378:metabolism*; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism*
chemical_list: D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D007461:Iodoacetates; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D006861:Hydrogen Peroxide; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; D010658:Phenylglyoxal; D019807:Iodoacetic Acid