Kinetic mechanism and intrinsic isotope effects for the peptidylglycine α-amidating enzyme reaction

Wilson A. Francisco, David J. Merkler, Ninian Blackburn, Judith P. Klinman

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Abstract

The bifunctional peptidylglycine α-amidating enzyme catalyzes the C- terminal amidation of glycine-extended peptides. The first enzyme activity, peptidylglycine α-hydroxylating monooxygenase, catalyzes the oxygen-, ascorbate-, and copper-dependent formation of α-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 carded 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 α-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 α-secondary deuterium intrinsic isotope effects, respectively. These values are identical to previous measurements for the analogous enzyme system, dopamine β-monooxygenase [Miller, S. M., and Klinman, J. P. (1985) Biochemistry 24, 2114-2127]. The identity of intrinsic isotope effects for peptidylglycine α-hydroxylating monooxygenase and dopamine β-monooxygenase with substrates of comparable reactivity (N- benzoylglycine and dopamine, respectively) extends similarities between the two enzymes significantly beyond sequence homology and cofactor requirements.

Original languageEnglish (US)
Pages (from-to)8244-8252
Number of pages9
JournalBiochemistry
Volume37
Issue number22
DOIs
StatePublished - Jun 2 1998

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Mixed Function Oxygenases
Isotopes
Kinetics
Enzymes
Dopamine
Enzyme activity
peptidylamidoglycolate lyase
Substrates
Oxygen
Peptides
Biochemistry
Lyases
Deuterium
Glycine
Sequence Homology
Labels
Copper
Derivatives
hippuric acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetic mechanism and intrinsic isotope effects for the peptidylglycine α-amidating enzyme reaction. / Francisco, Wilson A.; Merkler, David J.; Blackburn, Ninian; Klinman, Judith P.

In: Biochemistry, Vol. 37, No. 22, 02.06.1998, p. 8244-8252.

Research output: Contribution to journalArticle

Francisco, Wilson A. ; Merkler, David J. ; Blackburn, Ninian ; Klinman, Judith P. / Kinetic mechanism and intrinsic isotope effects for the peptidylglycine α-amidating enzyme reaction. In: Biochemistry. 1998 ; Vol. 37, No. 22. pp. 8244-8252.
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