The use of pH and kinetic isotope effects to probe the mechanism of D-amino acid oxidase

Abstract

The mechanism of hog kidney D-amino acid oxidase with glycine, D-alanine and D-serine as substrates has been examined in detail using the methods of pH effects and primary, secondary and solvent kinetic isotope effects. 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] of 10.7 be protonated. The DV/K[ser] value of 4.5 is pH-independent and intrinsic as confirmed by the primary deuterium isotope effect in the anaerobic rapid reaction and by the primary tritium isotope effect. With D-alanine, the pK[a] values are perturbed outwardly. The DV/Kaia value increases with decreasing pH, establishing that D-alanine is sticky. 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. The intrinsic DV/K[ala] value of 5.8 with D-alanine was determined from the TV/K[ala] value at low pH. With glycine, the pH optimum is shifted and the DV/K[gly] value increases with increasing pH, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At high pH, the intrinsic DV/K[gly] value is 3.6 which was confirmed by the isotope effect on the limiting rate of reduction and by the TV/K[gly] value. No significant a-secondary, 13-secondary or solvent Kinetic isotope effects were observed at the conditions where CH bond cleavage is totally rate-determining. These results argue against a concerted mechanism for reduction. A protonated group with a pK[a] value of 10.5 is important for the reaction of reduced flavin enzyme:imino acid complex with oxygen. Consistent with the bimolecular reaction of oxygen with reduced enzyme being rate-limiting, no solvent isotope effects were observed on the V/K[O2] value. Significant solvent isotope effects on turnover with D-alanine as substrate are consistent with a single slow proton transfer which mediates a conformational change, permitting imino acid release.