Mechanism of porphobilinogen synthase. Requirement of Zn2+ for enzyme activity

• Published in: The Journal of biological chemistry Vol. 255; no. 5; pp. 2030 - 2035
• Main Authors: Bevan, D R, Bodlaender, P, Shemin, D
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10.03.1980
• Subjects: Animals; Cattle; Dithiothreitol - pharmacology; Kinetics; Levulinic Acids; Liver - enzymology; Porphobilinogen Synthase - metabolism; Protein Binding; Spectrometry, Fluorescence; Zinc - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)85987-2

The role of metal ions in the mechanism of action of bovine liver porphobilinogen synthase was investigated. Studies with chelating agents were consistent with a requirement of metal ions for enzyme activity, and the use of 8-hydroxyquinoline-5-sulfonic acid suggested that Zn2+ was present in the enzyme. The low activity detected in metal-free apoporphobilinogen synthase was attributed to adventitious metal ions. Addition of Zn2+ to the apoenzyme completely restored enzyme activity if the essential sulfhydryl groups on the enzyme were first reduced with sulfhydryl reagents. It does not follow necessarily from this observation that Zn2+ forms a bond with a sulfhydryl group in the enzyme. However, we also observed that Zn2+ did not bind to the enzyme unless the essential cysteinyl residues were reduced. We have concluded that the octameric enzyme contains 4 g atoms of Zn2+/mol from our enzyme activity measurements and binding studies. Alkylation of the enzyme resulted in a marked reduction in the binding of Zn2+ to the enzyme. These observations are consistent with the suggestion that the interaction of the Zn2+ ions with the enzyme occurs with sulfhydryl groups at the active site. It appears that Zn2+ does not participate in substrate binding nor in the maintenance of the quaternary structure of the enzyme. Possible mechanistic roles for Zn2+ in porphobilinogen synthase are discussed. It should be noted that Cd2+ was the only other element found which restored activity to the apoenzyme.