Role of arginine 285 in the active site of Rhodotorula gracilis D-amino acid oxidase - A site-directed mutagenesis study

Arg(285), one of the very few conserved residues in the active site of D-am ino acid oxidases, has been mutated to lysine, glutamine, aspartate, and al anine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO), The mutat ed proteins are all catalytically competent. Mutations of Arg(285) result i n an increase ( approximate to 300-fold) of K-m for the D-amino acid and in a large decrease (approximate to 500-fold) of turnover number. Stopped-flo w analysis shows that the decrease in turnover is paralleled by a similar d ecrease in the rate of flavin reduction (k(2)), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg(285) in the mutate d DAAOs drastically reduces the binding of several carboxylic acids (e.g. b enzoate). These results highlight the importance of this active site residu e in the precise substrate orientation, a main factor in this redox reactio n. Furthermore, Arg(285) DAAO mutants have spectral properties similar to t hose of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme . From this, we can unexpectedly conclude that Arg(285) in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C( 2)=O locus of the isoalloxazine ring of the flavin. We also suggest that th e residue undergoes a conformational change in order to bind the carboxylat e portion of the substrate/ligand in the complexed enzyme.