pH and kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase.

Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pKa values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration (approximately 15 microM) at pH 9,D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKa values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of approximately 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H3O+ and H2O below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kgly value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.     

Crystallographic studies on D-amino acid oxidase.

D-amino acid oxidase, a flavoprotein from hog kidneys, has been crystalized in two different forms. Orthorhombic prisms have been obtained from the enzyme.benzoate complex at pH 8.3; the space group is C2221 and the cell dimensions are a = 325A, b = 138.8 A, c = 200 A. At lower pH values, the enzyme crystallizes in trigonal prisms with a = b = 116.0 A, c = 399 A, space group P3112 or its enantiomorph. The two crystal forms have been obtained at 28 degrees C while at 4 degrees C only weak evidence of crystallization has been detected. In both crystalline modifications, the protein is highly associated.     

Kinetic properties of rat kidney D-amino acid oxidase associated with peroxisomes

In contrast to hog kidney D-amino acid oxidase, the v vs s plots of D-amino acid oxidase in homogenized rat kidney did not have the form of a rectangular hyperbola, and showed an apparent negative cooperativity. After subcellular fractionation of rat kidney, both of the oxidases in the supernatant fraction and the peroxisomal fraction showed Michaelis-Menten type kinetics. The Km values for D-alanine and D-proline of the peroxisomal fraction were significantly lower than those of the supernatant fraction. The partially purified enzyme from the peroxisomal fraction showed the same kinetic properties as the supernatant fraction. These facts suggest that the two types of rat kidney D-amino acid oxidase were originally identical and that some interaction between the enzyme and peroxisomes is physiologically important for the function of the enzyme.