title: Metal binding properties of thiols; complexes with horse liver alcohol dehydrogenase.
abstract: The metal binding properties of thiols were investigated fluorimetrically and spectrophotometrically using horse liver alcohol dehydrogenase as a model metalloenzyme. The steady-state kinetics revealed that in the presence of the coenzyme the primary interaction of a thiol with the enzyme is by thiolate competing with alcohol for the active zinc site. The experiments with 2-mercaptoethanol and ethanethiol showed that at physiological pH it is enzyme-NAD-thiol complexes which are kinetically important with enzyme-thiol complexes only significant at higher pH. The dissociation constants for the binding of thiols in the ternary enzyme-NAD-thiol complexes showed tighter binding as the pH increases, with dithiols binding more tightly than monothiols. The primary binding to zinc was less dependent on the pKa value of each thiol than on mutual stabilization of zinc bound thiolate by the positive charge on the pyridinium ring of NAD, and by monodentate binding and with some dithiols perhaps bidentate binding. The tighter binding to the enzyme of the thiol when it is more hydrophobic or less polar indicates that the thiols interact not only with the active zinc site but also with a neighboring hydrophobic site. This is important for tight binding thiols which are rigidly held by multivalent binding through being anchored hydrophobically and to positively charged centers like zinc.
authors.affiliation: Institute of Biochemistry, University of Oslo, Norway.
authors.forename: B T
authors.initials: BT
authors.lastname: Langeland
authors.forename: D L
authors.initials: DL
authors.lastname: Morris
authors.forename: J S
authors.initials: JS
authors.lastname: McKinley-McKee
mesh_terms: D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000737:chemistry; D005470:Fluorometry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008623:Mercaptoethanol / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D009243:NAD / Q000737:chemistry; D011485:Protein Binding; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D013438:Sulfhydryl Compounds; D009243:NAD; D000431:Ethanol; D008623:Mercaptoethanol; D000426:Alcohol Dehydrogenase; D015032:Zinc; C007638:ethanethiol