STUDIES ON THE KINETIC MECHANISM OF PIG-KIDNEY D-AMINO-ACID OXIDASE BY SITE-DIRECTED MUTAGENESIS OF TYROSINE-224 AND TYROSINE-228

Expression conditions in Escherichia coli of wild-type, Y224F, and Y22 8F mutants of pig kidney D-amino acid oxidase (DAAO) have been changed to yield more enzyme. The mutated proteins show spectral properties s imilar to those of the wild-type enzyme, in all oxidation-reduction st ates. Ah enzymes were studied by steady state and rapid reaction metho ds. Turnover numbers determined for Y224F DAAO with different substrat es were similar to those of wild-type protein, while the Y228F DAAO al ways showed lower turnover numbers and higher K-m values for the D-ami no acid. Analyses of reduction traces at 450 and 550 nm of stopped-flo w experiments with wild-type DAAO showed the presence of a new phase, the conversion between two different charge-transfer complexes of the reduced enzyme and imino acid product. The substitution of Tyr-228 tot ally abolished the formation of the long wavelength bands while Y224F DAAO showed long wavelength absorbance only for the first intermediate . Reoxidation of the reduced flavin results from reaction of oxygen wi th the first charge-transfer complex. The rate of reduction with D-ala nine as substrate was 1225,45 and 10 s(-1) for wild-type, Y224F, and Y 228F DAAOs, respectively. Comparison of the properties of these two mu tant enzyme forms with those of the wild-type DAAO indicate that both tyrosine residues have their main function in the reductive half-react ion of the; enzyme.