Abstract
The mechanism of D-amino acid oxidase has been examined using nitroalkane anions, viscosity effects, mutant proteins, and ¹⁵N kinetic isotope effects. From the studies on the effects of pH on the V/K[] values for the nitroalkane anions, a group with a pK[] value of 6.8 must be unprotonated and that a group with a pK[] value of 9.7 must be protonated for catalysis. The []V/K[] value of 0.84 is pH-independent, and the results are consistent that the oxidation of nitroalkane anions by D-amino acid oxidase proceeds by a direct attack of the carnation on the N(5) position of the flavin. The V/K value for D-alanine is insensitive to solution viscosity. This indicates the reaction of D-alanine with DAAO is not limited by diffusion. The insensitivity to solution viscosity could be interpreted as evidence that there is a slow conformational change after formation of the initial enzyme and substrate complex but before any chemical steps occur. The roles of H217 and H307 in catalysis were examined by substitution of these residues. The kinetic parameters for the H307S mutant protein were essentially identical to those of wild-typd enzyme. The H217A mutant protein was active when compared to wild-type enzyme. These results suggest that the H307 and H217 residues do not have essential roles in catalysis. The mechanism of cleavage of the CH bond of the amino acid substrate by D-amino acid oxidase was investigated by determining the ¹⁵N kinetic isotope effects with D-alanine and D-serine as substrates. The measured []V/K[] isotope effects decrease at higher pH and increase in D₂O suggesting that the unprotonated form of the amino group is the substrate for D-amino acid oxidase. However, the results are inconclusive due to poor precision.
Kurtz, Kevin Anthony (2000). Studies on the mechanism of D-amino acid oxidase. Master's thesis, Texas A&M University. Available electronically from
https : / /hdl .handle .net /1969 .1 /ETD -TAMU -2000 -THESIS -K87.