INTERACTION OF PROTOCATECHUATE-3,4-DIOXYGENASE WITH FLUORO-SUBSTITUTED HYDROXYBENZOIC ACIDS AND RELATED COMPOUNDS

The substrate analogues 3-fluoro-4-hydroxybenzoic acid (3-FHB) and 4-fluoro-3-hydroxybenzoic acid (4-FHB) were synthesized and examined as active site probes for protocatechuate-3,4-dioxygenase (PCD). Upon incubation of 3-FHB with PCD, a complex is formed which causes alterations in both the visible and CD spectra of the enzyme, and these changes can be reversed by dialysis or by displacement with other ligands. Upon prolonged incubation of 3-FHB with PCD, neither oxidation of the fluoro compound nor irreversible inactivation of the enzyme occurs. Steady-state kinetic measurements established that 3-FHB acts as a simple competitive inhibitor of PCD. Inhibition constants were determined in the pH range 6.0 to 9.4, with maximal potency at pH 7.0 where 3-FHB is the most potent PCD inhibitor known (K1 = 0.3 μM). 3-FHB is an ideal active site titrant for PCD, and titration data indicate that 8.0 mol of ligand bind per mol of enzyme. Mathematical analysis of the titration data gave dissociation constants in excellent agreement with the kinetically determined K1 values at several different pHs. In contrast, the isomeric 4-FHB was found to be a very weak PCD inhibitor with a K1 approximately 1000-fold greater than that of 3-FHB. The contrasting behavior of the isomeric fluorohydroxybenzoates was found to be mirrored in the behavior of the corresponding defluoro ligands, 3- and 4-hydroxybenzoic acid (3-HB and 4-HB, respectively), but the presence of the fluorine substituent in 3-FHB increases the ligand's affinity for the enzyme approximately 500-fold. Analysis of the pH dependencies of inhibition constants indicated preferential binding of the protonated forms of 3-FHB and 4-HB. That the efficient binding of 3-FHB at the active site cannot be attributed solely to simple chelation of the iron atom is supported by spectral examination of Fe (III) complexation by 3-FHB and related compounds. Displacement studies were carried out using stopped-flow techniques in order to examine the kinetics of dissociation of the complexes of PCD with 3-FHB and other inhibitors. The utility of fluoro substituted ligands as active site probes for analysis of binding interactions and for evaluation of catalytic mechanisms for dioxygenases such as PCD is discussed. © 1978, American Chemical Society. All rights reserved.