title: Active site histidine in pig liver aminolevulic acid dehydratase modified by diethylpyrocarbonate and protected by Zn2+ ions.
abstract: 1. The effect of diethylpyrocarbonate (DEP) (0.1-0.35 mM) on the purified pig liver amino-levulic acid dehydratase (ALA-D) containing 0.3 g-atoms Zn/subunit, under different pHs (6.0-7.5), temperature (0-18 degrees C) and time (0-60 min) was studied. 2. Three histidyl residues/subunit were modified by DEP (0.2 mM, pH 6.8), but activity was completely lost after the first one had reacted, indicating the presence of one histidine residue essential for ALA-D catalysis. Reactivation by treatment with hydroxylamine (0.7 mM, pH 7.0) confirmed that only histidine and no other nucleophile amino acids were directly involved in DEP inhibition. 3. Zn ions (0.5 mM) and the substrate ALA (5-10 mM) protected against DEP inactivation, protection was dependent on pH. 4. Sn, Se, Hg, Cd, Mn, Co and Pb (0.01-0.1 mM) did not significantly protect ALA-D against inactivation. 5. It is concluded that the substrate and Zn binding sites and the essential histidyl residues are in close proximity in the active center. It is proposed that in the catalytic synthesis of porphobilinogen from ALA, histidine groups have the specific role of transporting protons from the aqueous media to a hydrophobic active site.
authors.affiliation: Centro de Investigaciones sobre Porfirinas y Porfirias-CIPYP (CONICET-FCEyN, UBA), Ciudad Universitaria, Buenos Aires, Argentine.
authors.forename: H
authors.initials: H
authors.lastname: Fukuda
authors.forename: S R
authors.initials: SR
authors.lastname: Paredes
authors.forename: A M
authors.initials: AM
authors.lastname: Batlle
mesh_terms: D000818:Animals; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008670:Metals / Q000494:pharmacology; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013552:Swine; D015032:Zinc / Q000494:pharmacology
chemical_list: D008670:Metals; D006639:Histidine; D000623:Porphobilinogen Synthase; D015032:Zinc; D004047:Diethyl Pyrocarbonate