The alpha-amidating enzyme. The intermediary reaction steps and the structure/function relationship as a bifunctional enzyme.

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Peptidylglycine alpha-amidating activity catalyzes the oxidation of a C-terminally glycine-extended peptide to a desglycine alpha-amidated peptide at the expense of ascorbate and O_2 in the presence of copper. The reaction involves oxidative N-dealkylation within the terminal glycine, retaining the glycine N and releasing the remainder as glyoxylate. We revealed that the reaction consists of two steps via a carbinolamide as an intermediate (peptidyI alpha-hydroxyglycine), and also that two separate enzymes derived from a common precursor protein, peptidylglycine alpha-hydroxylase (PH) and peptidylamidoglycolate lyase (PGL), catalyze these steps, formation of the carbinolamide and its conversion into the alpha-amide and glyoxylate, respectively. The goals of the projects were (1) to clarify whether an alpha-carbon radical of glycine is involved in the formation of the carbinolamide intermediate by investigating isotope effects on the PH reaction, and (2) to construct an expression vecto … More r that is able to secrete the precursor protein into medium in order to probe the structure/function relationship as a bifunctional enzyme. Along with these projects, we have obtained the following results. (1) The hydroxylation of glycine is a ping-pong Bi Bi reaction in terms of ascorbate and the substrate (D-Tyr-Val-Gly). The deuterium effects on V/K and Vmax, using D-Tyr-Val-Gly-2,2-d2, are about 1.2, far less than 5.7 which has been reported by Kizar et al. Much more detailed kinetic study with tritium-labeled substrate is on going in order to learn the true isotope effect values, ^DV/K and ^DVmax. (2) The cDNA library has been prepared from rat atrium. The cDNA clone encoding the precursor protein is in search. (3) We have found the amidating activity in the cerebrospinal fluids (CSF) is increased in patients with multiple sclerosis, especially during the active stage, compared with that in patients with other neurological diseases. This suggests that the amidating activity in CSF could be an indication reflecting the physiological and morbid states of the central nervous system. Less