| Abstract: | The presence of arginine in the active center of D-amino-acid oxidase is well documented although its role has been differently interpreted as being part of the substrate-binding site or the positively charged residue near the N1-C2 = O locus of the flavin coenzyme. To have a better insight into the role of the guanidinium group in D-amino-acid oxidase we have carried out inactivation studies using phenylglyoxal as an arginine-directed reagent. Loss of catalytic activity followed pseudo-first-order kinetics for the apoprotein whereas the holoenzyme showed a biphasic inactivation pattern. Benzoate had no effect on holoenzyme inactivation by phenylglyoxal and the coenzyme analog 8-mercapto-FAD did not provide any additional protection in comparison to the native coenzyme. Spectroscopic experiments indicated that the modified protein is unable to undergo catalysis owing to the loss of coenzyme-binding ability. Analyses of time-dependent activity loss versus arginine modification or 14C]phenylglyoxal incorporation showed the presence of one arginine essential for catalysis. The protection exerted by the coenzyme is consistent with the involvement of an active-site arginine in the correct binding of FAD to the protein moiety. Comparative analyses of CNBr fragments obtained from apoenzyme, holoenzyme and the 8-mercapto derivative of D-amino-acid oxidase after reaction with phenylglyoxal did not provide unequivocal identification of the essential arginine residue within the primary structure of the enzyme. However, they suggest that it might be localized in the N-terminal portion of the polypeptide chain and point to a role of phenylglyoxal-modifiable arginine in binding to the adenylate/pyrophosphate moiety of the flavin coenzyme. |
