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.
Denu
J M
JM
Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843.
Fitzpatrick
P F
PF
D000409:Alanine / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D003903:Deuterium; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D012694:Serine / Q000378:metabolism
D012694:Serine; D003903:Deuterium; D003605:D-Amino-Acid Oxidase; D000409:Alanine; D005998:Glycine