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 Y228F mutants of pig kidney D-amino acid oxidase (DAAO) have been changed to yield more enzyme. The mutated proteins show spectral properties similar to those of the wild-type enzyme, in all oxidation-reduction states. All enzymes were studied by steady state and rapid reaction methods. Turnover numbers determined for Y224F DAAO with different substrates were similar to those of wild-type protein, while the Y228F DAAO always showed lower turnover numbers and higher Km values for the D-amino acid. Analyses of reduction traces at 450 and 550 nm of stopped-flow 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 totally 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 with the first charge-transfer complex. The rate of reduction with D-alanine as substrate was 1225,45 and 10 s-1 for wild-type, Y224F, and Y228F DAAOs, respectively. Comparison of the properties of these two mutant enzyme forms with those of the wild-type DAAO indicate that both tyrosine residues have their main function in the reductive half-reaction of the enzyme.
Pollegioni
L
L
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Fukui
K
K
Massey
V
V
D000818:Animals; D002384:Catalysis; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013447:Sulfites / Q000378:metabolism; D013552:Swine; D014443:Tyrosine / Q000737:chemistry; D062367:ortho-Aminobenzoates / Q000378:metabolism
D013447:Sulfites; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D014443:Tyrosine; D003605:D-Amino-Acid Oxidase