Kinetics of pH-dependent interconversion of tryptophanase spectral forms studied by scanning stopped-flow...

Morino and Snell [Morino, Y., & Snell, E. E. (1967) J. Biol. Chem. 242, 5591-5601] previously showed that the relative amplitudes of the 337- and 420-nm absorption bands of tryptophanase depended on both pH and the nature of a required monovalent cation activator. An investigation of the kinetics of interconversion of the 337- and 420-nm forms following a rapid incremental increase (jump) or decrease (drop) in pH over the range of enzyme stability in 0.2 M KCl at 24 +/- 0.3 degrees C by scanning stopped-flow spectrophotometry showed three distinct time-dependent phases. They were (1) an abrupt phase which is complete in less than 6.5 ms, (2) a fast first-order interconversion of the 420- and 337-nm absorbances, and (3) a slow first-order process involving growth at 355 nm coupled to two decays centered at 325 and 430 nm in the incremental pH jumps and decay at 355 nm with concomitant growth at 430 and 290 nm in the incremental pH-drop experiments. The results of these experiments were analyzed in terms of a scheme involving enzyme forms E alpha, E beta, E beta H+, E gamma, E gamma H+, and E delta. The E alpha form predominates in the absence of activating monovalent cations and absorbs at 420 nm. Those in the beta manifold, E beta and E beta H+, also absorb at 420 nm while those in the gamma manifold, E gamma and E gamma H+, absorb at 337 nm. The form E delta absorbs at 335 nm. E beta H+ and E gamma H+ represent the protonated form of the enzyme in each manifold. Analysis of the abrupt phase showed no significant systematic changes in absorbance above 330 nm for either the pH-jump or pH-drop experiments. The fast second phase involves the first-order interconversion of the beta and gamma manifolds while the slow third phase describes the buildup or decay of the delta manifold. Presumably conformational changes control the rate of these interconversions. The pH dependence of the fast first-order beta to gamma conversion was described and evaluated in terms of five independent equilibrium and rate constants and three independent amplitude terms by simultaneously fitting the amplitude data and first-order rate constants to an equation describing the overall scheme with a nonlinear least-squares program KINFIT4 [Dye, J. L., & Nicely, V. A. (1971) J. Chem. Educ. 48, 443-448]. The pK for protonation of the beta form = 9.70 +/- 0.12, for protonation of the gamma form (337-nm absorber) = 6.77 +/- 0.10, and for the pH-dependent interconversion of the beta and gamma manifolds, pKa = 8.11 +/- 0.04. The computed equilibrium distribution among the four species of the beta and gamma manifolds showed that E beta H+ and E gamma predominate.