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Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex.

Abstract：

:The reaction of D-amino acid oxidase [EC 1.4.3.3] (DAO) from porcine kidney with beta-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-BCNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of beta-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as alpha-amino-beta-cyanoacrylate and the charge transfer complex as the complex of alpha-amino-alpha-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present finding is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-benzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Miura R,Shiga K,Miyake Y,Watari H,Yamano T

doi

10.1093/oxfordjournals.jbchem.a132888

subject

Has Abstract

pub_date

1980-05-01 00:00:00

pages

1469-81

issue

eissn

0021-924X

issn

1756-2651

journal_volume

pub_type

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Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex.