Effect of pH on the interaction of benzoate and D-amino acid oxidase

Abstract

The kinetic and equilibrium Kd of the reversible binding of benzoate to hog kidney D-amino acid oxidase (DAAO) were studied at 19.degree. C over the pH range 5.3-10.5 by means of a stopped-flow apparatus and spectrophotometric titrations. A simple bimolecular reaction of the form 2nd order-1st order was observed; a 2-step reaction was not seen. Analysis of the pH dependence of the bimolecular rate constants and equilibrium Kd is consistent with 3 ionizable groups which are important for benzoate binding. The pK values of the enzyme-related ionizations are 6.3, 9.2 and 9.6. Analysis of the change in extinction coefficient at 360 nm indicates the pk of 9.6 can be assigned to be 3-imino group of the enzyme-bound flavin. The effect of benzoate on the apparent pk for the ionization of the 3-imino group of the enzyme-bound FAD was reexamined. The presence of benzoate causes an apparent shift of this ionization from a pK value of 9.6 to 10.7.