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.