PROTOCATECHUATE 3,4-DIOXYGENASE - IMPLICATIONS OF IONIZATION EFFECTS ON BINDING AND DISSOCIATION OF HALOHYDROXYBENZOATES AND ON CATALYTIC TURNOVER

The effect of pH upon the binding and dissociation of halohydroxybenzoate inhibitors of protocatechuate 3,4-dioxygenase (PCD) and on catalytic turnover with the substrates protocatechuic acid (PCA) and 3,4-dihydroxyphenylpropionic acid (DHPP) has been investigated. The kcat profile for PCA shows a simple sigmoidal pH dependence, with an apparent pof 7.0 ±0.1. The kcat/Km profile obtained in phosphate exhibits an apparent pKa of ~7, while that obtained in zwitterionic buffers shows only slight variation in the pH region from 5.5 to 7.5 and a decrease above pH 8. The pH dependence of breakdown of the E-S-O2 complex, formed from the slow substrate, DHPP, also exhibits a sigmoidal variation with pH, with an apparent pKa of 8.0 ±0.1. The pH dependence of binding of the potent inhibitors 3-fluoro-4-hydroxybenzoic acid and 3-chloro-4-hydroxybenzoic acid demonstrates that the strong binding of these compounds is not attributable to the increased acidity of the phenolic moiety. A linear freeeneryplot of the 3-halo-4-hydroxybenzoates reveals that steric factors are important in determining the relative potency of these inhibitors. To obtain further information on the interaction of PCD with the inhibitors, we measured the dissociation rates via stopped-flow displacement. These dissociation rates show a dramatic increase at alkaline pH values, which parallels the rise in both K1 and Km. In contrast, the pH dependence of 4-nitrocatechol (4-NC) dissociation is bell-shaped and decreases at alkaline pH. The kinetics of association of 4-NC with PCD were analyzed and are consistent with a rapid ligation step followed by a slow process which yields the characteristic 550-nm peak of the PCD-4-NC complex. With regard to catalytic turnover, evidence has been obtained that the Kcat profile reflects an ionization which directly facilitates breakdown of the E-S-O2 complex. The implications of these findings are discussed in terms of the structure of the E-S-O2 complex and the mechanism of PCD catalysis. © 1979, American Chemical Society. All rights reserved.