Involvement of metals in enzymatic and nonenzymatic decomposition of C-terminal alpha-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 alpha-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL); PHM catalyzes the hydroxylation of C-terminal glycine, and PAL decomposes the peptidyl-alpha-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, Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), 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; k(cat) 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, Zn(2+), Co(2+), and Cd(2+) 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 k(cat) increased 3- and 5-fold and the K(m) 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.
Takahashi
Kenichi
K
Department of Medical Biochemistry, Kurume University School of Medicine, Kurume 830-0011, Japan.
Harada
Saori
S
Higashimoto
Yuichiro
Y
Shimokawa
Chizu
C
Sato
Hideaki
H
Sugishima
Masakazu
M
Kaida
Yasuhiko
Y
Noguchi
Masato
M
D000577:Amides / Q000378:metabolism*; D019760:Amidine-Lyases / Q000378:metabolism*; D055162:Biocatalysis; D005998:Glycine / Q000378:metabolism*; D015032:Zinc / Q000502:physiology*
D000577:Amides; D019760:Amidine-Lyases; C068521:peptidylamidoglycolate lyase; D015032:Zinc; D005998:Glycine