Involvement of Metals in Enzymatic and Nonenzymatic Decomposition of C-Terminal α-Hydroxyglycine to Amide: An Implication for the Catalytic Role of Enzyme-Bound Zinc in the Peptidylamidoglycolate Lyase Reaction

The peptide C-terminal amide group essential for the full biological activity of many peptide hormones is produced by consecutive actions of peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL); PHM catalyzes the hydroxylation of C-terminal glycine, and PAL decomposes the peptidyl-α-hydroxyglycine to an amidated peptide and glyoxylate. PAL contains 1 mol of zinc, but its role, catalytic or structural, has not yet been clarified. In this study, we found that a series of transition metals, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+, catalyze the nonenzymatic decomposition of the hydroxyglycine intermediate in a concentration-dependent manner. The second-order rate constant of the metal catalysis increased with elevation of pH, indicating that the hydrated metal acts as a general base. Extensive removal of the enzyme-bound metals remarkably diminished the PAL activity; kcat of the metal-depleted enzyme retaining 0.1 mol of zinc decreased to 3.2 s−1 from 25.7 s−1 of the wild-type enzyme. Among a series of divalent metals tested, Zn2+, Co2+, and Cd2+ could fully restore the PAL activity of the metal-depleted enzyme. Especially, Zn substitution reproduced the steady-state parameters of the wild-type enzyme. On the other hand, Co and Cd substitution largely altered the kinetic parameters; the kcat increased 3- and 5-fold and the Km for the substrate increased 2.5- and 4-fold, respectively. These observations support that the enzyme-bound zinc plays a catalytic role, rather than a structural role, in the PAL reaction through the action of zinc-bound water as a general base.