title: Metal ion selectivity and substrate inhibition in the metal ion chelation catalyzed by human ferrochelatase.
abstract: Protoporphyrin IX ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the k(cat)/K(m)(app) ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent k(cat) depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnD(IX)) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc.
authors.affiliation: Department of Chemistry, University of Sheffield, Sheffield S3 7HF, United Kingdom.
authors.forename: Ruth E
authors.initials: RE
authors.lastname: Davidson
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Chesters
authors.forename: James D
authors.initials: JD
authors.lastname: Reid
mesh_terms: D002384:Catalysis; D002614:Chelating Agents / Q000737:chemistry / Q000494:pharmacology*; D003902:Detergents / Q000737:chemistry; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005294:Ferrochelatase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000737:chemistry*; D008956:Models, Chemical; D015233:Models, Statistical; D011166:Porphyrins / Q000737:chemistry; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry
chemical_list: D002614:Chelating Agents; D003902:Detergents; D004791:Enzyme Inhibitors; D007477:Ions; D008670:Metals; D011166:Porphyrins; D005294:Ferrochelatase; D015032:Zinc