Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex.
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.
D000409:Alanine / Q000031:analogs & derivatives*; D000818:Animals; D002942:Circular Dichroism; D003486:Cyanides; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D010101:Oxygen Consumption; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013552:Swine
D003486:Cyanides; C004631:3-cyanoalanine; D003605:D-Amino-Acid Oxidase; D000409:Alanine