CHEMICAL MECHANISM OF D-AMINO-ACID OXIDASE FROM RHODOTORULA-GRACILIS - PH-DEPENDENCE OF KINETIC-PARAMETERS

The variation of kinetic parameters of D-amino acid oxidase from Rhodi otorula gracilis with pH was used to gain information about the chemic al mechanism of the oxidation of D-amino acids catalysed by this flavo enzyme. D-Alanine was the substrate used. The pH dependence of V-max a nd V-max/K-m for alanine as substrate showed that a group with a pK va lue of 6.26-7.95 (pK(1)) must be unprotonated and a group with a pK of 10.8-9.90 (pK(2)) must be protonated for activity. The lower pK value corresponded to a group on the enzyme involved in catalysis and whose protonation state was not important for binding. The higher pK value was assumed to be the amino group of the substrate. Profies of pK(1) f or D-aspartate as competitive inhibitor showed that binding is prevent ed when a group on the enzyme with a pK value of 8.4 becomes unprotona ted; this basic group was not detected in V-max/K-m profiles suggestin g its involvement in binding of the beta-carboxylic group of the inhib itor.