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

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Abstract
Morino and Snell previously showed that the relative amplitudes of the 337 and 420 nm absorption bands of Escherichia coli tryptophanase depended on pH and on 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.degree. C by scanning stopped-flow spectrophotometry showed 3 distinct time-dependent phases. They were an abrupt phase which is complete in < 6.5 ms, a fast 1st-order interconversion of the 420 and 337 cm absorbances and a slow 1st-order process involving growth at 355 nm coupled to 2 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 355 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 changes in absorbance above 330 nm for the pH-jump on the pH-drop experiments. The fast 2nd phase involves the 1st-order interconversion of the .beta. and .gamma. manifolds while the slow 3rd 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 1st-order .beta. to .gamma. conversion was described and evaluated in terms of 5 independent equilibrium and rate constants and 3 independent amplitude terms by simultaneously fitting the amplitude data and 1st-order rate constants to an equation describing the overall scheme with a nonlinear least-squares program KINFIT4 [Dye and Nicely (1971)]. 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, pK.alpha. = 8.11 .+-. 0.04. The computed equlibrium distribution among the 4 spp. of the .beta. and .gamma. manifolds showed that E.beta.H+ and E.gamma. predominate.
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