1998
DOI: 10.1021/bi973004y
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Kinetic Mechanism and Intrinsic Isotope Effects for the Peptidylglycine α-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 iso… Show more

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Cited by 91 publications
(167 citation statements)
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“…This is consistent with the small substrate H/D kinetic isotope effect on k cat (1.5) (9), where the calculated H/D isotope effect on this rebound step is 1.1. Furthermore, the 16=18 O 2 isotope effect on rebound is calculated to be 1.023, similar to the observed 16=18 O 2 isotope effect on k cat /K m (1.017) (8). The overall calculated barrier of ΔG ‡ = +23.8 kcal/mol [i.e., from Cu I M ðOH 2 Þ to the rebound TS] is somewhat higher than that measured in turnover [∼17 kcal/mol, calculated from k cat = 39 s -1 at 37°C (8)]; however, this difference can be attributed to the constraints imposed on the active site in the computational model.…”
Section: Discussionsupporting
confidence: 76%
“…This is consistent with the small substrate H/D kinetic isotope effect on k cat (1.5) (9), where the calculated H/D isotope effect on this rebound step is 1.1. Furthermore, the 16=18 O 2 isotope effect on rebound is calculated to be 1.023, similar to the observed 16=18 O 2 isotope effect on k cat /K m (1.017) (8). The overall calculated barrier of ΔG ‡ = +23.8 kcal/mol [i.e., from Cu I M ðOH 2 Þ to the rebound TS] is somewhat higher than that measured in turnover [∼17 kcal/mol, calculated from k cat = 39 s -1 at 37°C (8)]; however, this difference can be attributed to the constraints imposed on the active site in the computational model.…”
Section: Discussionsupporting
confidence: 76%
“…Also shown in Table 1 are the magnitudes of observed O-18 kinetic isotope effects using either H-or D-labeled substrates (14,17). Although the measured values differ somewhat between D␤M and PHM, consistent with somewhat different rate-limiting steps under steady state conditions, in both instances the magnitude of k 16 /k 18 is seen to increase when substrate is deuterated.…”
Section: Solution Studies Indicate That D␤m and Phm Are Mechanisticalmentioning
confidence: 72%
“…An isotope effect on k cat of ϳ2.7 indicates that hydrogen abstraction from the substrate is partially rate-limiting, as demonstrated for D␤M and PHM (23,24). Values for D k cat / K m,tyr appear to be virtually independent of ascorbate, which again points to an irreversible step separating reduction of the enzyme and the subsequent chemistry.…”
Section: Discussionmentioning
confidence: 85%
“…This fact and the comparatively low observed k cat value (D␤M ϭ 12.7 Ϯ 0.5 s Ϫ1 (dopamine); PHM ϭ 39.1 Ϯ 0.5 s Ϫ1 (hippuric acid) (18,23)) prompted us to investigate the ascorbate dependence on the T␤M reaction rates. Initial rates of oxygen consumption by the enzyme were measured as a function of tyramine, at fixed ascorbate concentrations ranging from 2 to 200 mM ( Fig.…”
Section: Resultsmentioning
confidence: 99%