THE CATALYTIC MECHANISM OF KYNURENINASE FROM PSEUDOMONAS-FLUORESCENS - INSIGHTS FROM THE EFFECTS OF PH AND ISOTOPIC-SUBSTITUTION ON STEADY-STATE AND PRE-STEADY-STATE KINETICS

The effects of pH and isotopic substitution of substrate and solvent o n the reaction of kynureninase from Pseudomonas fluorescens have been determined. The pH dependence of k(cat)/K-m for L-kynurenine is bell-s haped, with apparent pK(a)'s of 6.25 +/- 0.05 on the acidic limb and 8 .9 +/- 0.1 on the basic limb, and with a pH-dependent value of k(cat)/ K-m of 2 x 10(5) M-1 s(-1). The pH dependence of k(cat)/K-m for 3-hydr oxykynurenine is also bell-shaped, with apparent pK(a)'s of 6.49 +/- 0 .07 and 8.55 +/- 0.09, and with a pH-dependent value of 2.5 x 10(3) M- 1 s(-1). The k(cat) for L-kynurenine decreases at acidic pH values, wi th an apparent pK(a) of 6.43 +/- 0.06 and a pH-dependent value of 7 s( -1). The solvent kinetic isotope effect on k(cat) for the reaction of kynurenine in [H-2]H2O is 6.56 +/- 0.59, whereas there is no normal ki netic isotope effect on k(cat)/K-m, at pH 8.1. The proton inventory of k(cat) fits very well to the Gross-Butler equation, with x = 0.825 +/ - 0.08, suggesting that only a single proton is transferred in the rat e-determining step. In contrast, there is no significant kinetic isoto pe effect on either k(cat) or k(cat)/K-m with alpha-[H-2]-L-kynurenine as the substrate. There is a ''burst'' of anthranilate (0.7 mol/mol o f enzyme) formed in the pre steady state of the reaction of kynurenina se, with a rate constant of 54 s(-1) which is not affected by [H-2]H2O . The partition ratio of alanine to pyruvate formation is 2.3 x 10(4) in H2O and 6.9 x 10(3) in [H-2]H2O. Taken together, these data indicat e that the rate-limiting step in the reaction of kynureninase occurs s ubsequent to the first irreversible step, which is anthranilate releas e, is general base catalyzed, and involves transfer of only a single p roton. On the basis of these observations, we propose that the rate-li miting step in the reaction of kynureninase is C-4' deprotonation of t he pyruvate pyridoxamine 5'-phosphate ketimine intermediate.