ELECTROSTATIC EFFECTS ON SUBSTRATE ACTIVATION IN PARA-HYDROXYBENZOATEHYDROXYLASE - STUDIES OF THE MUTANT LYSINE-297 METHIONINE

p-Hydroxybenzoate hydroxylase (EC 1.14.13.2) is a flavoprotein monooxy genase that catalyzes the incorporation of one atom of molecular oxyge n into p-hydroxybenzoate to form 3,4-dihydroxybenzoate. The enzyme act ivates the substrate at the 3 position to electrophilic substitution b y lowering the pK(a) of the phenolic oxygen. The results presented her e indicate that regions of positive potential in the active site facil itate this substrate activation, which is necessary for rapid hydroxyl ation. We have neutralized a positive point charge by mutating lysine 297 to methionine (K297M). This mutation changes an amino acid near th e active site, but not directly in contact with the flavin or the subs trate, A variety of transient state kinetic and static parameters have been determined with two substrates. The results indicate that the K2 97M mutant does not activate the substrate through phenolic ionization to the same extent as wildtype (WT) and yet remains a competent hydro xylase. However, catalysis by the mutant is slow compared to that of W T, particularly in the oxidative half-reaction. Thus, normally quite l abile oxygenated flavin intermediates encountered in the hydroxylation pathway of WT p-hydroxybenzoate hydroxylase are stabilized and their decay is rate limiting in the K297M turnover, Electrostatic potential calculations offer an explanation for the lack of substrate activation . The stability of the oxidative reaction intermediates seems to be re lated to a lower degree of substrate activation.