title: Kinetic mechanism and intrinsic isotope effects for the peptidylglycine alpha-amidating enzyme reaction.
abstract: The bifunctional peptidylglycine alpha-amidating enzyme catalyzes the C-terminal amidation of glycine-extended peptides. The first enzyme activity, peptidylglycine alpha-hydroxylating monooxygenase, catalyzes the oxygen-, ascorbate-, and copper-dependent formation of alpha-hydroxyglycine derivatives. These are substrates for the second enzyme activity, peptidylamidoglycolate lyase, which catalyzes their breakdown to the corresponding C-terminal amidated peptide and glyoxylate as final products. Kinetic and isotope effect studies were carried out with N-benzoylglycine as a substrate at pH 6.0 using monofunctional and bifunctional monooxygenase activities. Kinetic data indicate an equilibrium ordered mechanism, with hippuric acid binding first followed by oxygen. A potentially important difference between the two monooxygenase activities is that product release occurs more slowly from the bifunctional enzyme, indicating an influence of the lyase domain on release of alpha-hydroxyglycine product to solution. Intrinsic isotope effects for the C-H bond cleavage were measured for the monofunctional form of the enzyme using a double-label tracer method, yielding 10.6 +/- 0.8 and 1.20 +/- 0.03 for the primary and alpha-secondary deuterium intrinsic isotope effects, respectively. These values are identical to previous measurements for the analogous enzyme system, dopamine beta-monooxygenase [Miller, S. M., and Klinman, J. P. (1985) Biochemistry 24, 2114-2127]. The identity of intrinsic isotope effects for peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase with substrates of comparable reactivity (N-benzoylglycine and dopamine, respectively) extends similarities between the two enzymes significantly beyond sequence homology and cofactor requirements.
authors.affiliation: Department of Chemistry, University of California, Berkeley 94720-1460, USA.
authors.forename: W A
authors.initials: WA
authors.lastname: Francisco
authors.forename: D J
authors.initials: DJ
authors.lastname: Merkler
authors.forename: N J
authors.initials: NJ
authors.lastname: Blackburn
authors.forename: J P
authors.initials: JP
authors.lastname: Klinman
mesh_terms: D001667:Binding, Competitive; D002384:Catalysis; D003903:Deuterium; D004299:Dopamine beta-Hydroxylase / Q000737:chemistry; D005998:Glycine / Q000737:chemistry*; D006626:Hippurates / Q000737:chemistry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D009097:Multienzyme Complexes; D013379:Substrate Specificity; D014316:Tritium
chemical_list: D006626:Hippurates; D009097:Multienzyme Complexes; D014316:Tritium; D003903:Deuterium; D006899:Mixed Function Oxygenases; D004299:Dopamine beta-Hydroxylase; C035189:peptidylglycine monooxygenase; C030514:hippuric acid; D005998:Glycine