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 pK_a value of 8.7 be unprotonated and that a group with a pK_a value of 10.7 be protonated. The ^DV/K_ser value of 4.5 is pH-independent, establishing that these pK₂ 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 15 /µM) 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 pK_ala values are perturbed to 8.1 and 11.5. The ^DV/K_ala 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 ~ 10. The effect of pH on the ^DV/K_ala value is consistent with a model in which exchange with solvent of the proton from the group with pK_a 8.7 is hindered and is catalyzed by H₂O and OH^- above pH 7 and by H₃0⁺ and H₂0 below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The ^DV/K_ala 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.