The pH-rate profiles for the kinetic parameters of 4-oxalocrotonate tautomerase (4-OT) have been measured using 2-hydroxy-2,4-hexadiendioate (2a) and 2-hydroxy-2,4-pentadienoate (2b) as substrates. The PH dependences of log (k(cat)/K-m) and of log k(cat) for the slow, nonsticky substrate 2b, which lacks a 6-carboxyl group, were bell-shaped with limiting slopes of unity on both sides of the PH optimum. For 2b, pK(a) values of 6.2 +/- 0.3 and 9.0 +/- 0.3 for the free enzyme (pK(E)) and 7.7 +/- 0.3 and 8.5 +/- 0.3 for the ES complex (pK(ES)) were obtained. The pK(E) of 6.2 +/- 0.3 for 2b represents a true pK, for a basic group on the enzyme and is most likely Pro-1 on the basis of inhibition studies with the substrate-based affinity label 3-bromopyruvate (3-BP) [Stivers et al. (1996) Biochemistry 35, 803-813]. Accordingly, N-15 NMR titration of the uniformly N-15-labeled enzyme showed that the pK(a) of the amino group of Pro-1 is 6.4 +/- 0.2, in reasonable agreement with those found by the effect of pH on k(cat)/K-m for 2b (6.2 +/- 0.3) and on k(inact)/K-I for 3-BP (6.7 +/- 0.3), but three units lower than the pK(a) of the model compound proline amide (pK(a) = 9.4 +/- 0.2), The pK(a) values for the two histidine residues of 4-OT, which were measured by H-1 NMR (His-6, pK(a) less than or equal to 5; His-49, pK(a) = 5.2 +/- 0.2), are at least one pK unit lower than the pK(E), excluding these residues as candidates for the general base. A plot of log (k(cat)/K-m) vs PH for the 10(4)-fold more reactive, but sticky substrate 2a [(k(cat)/K-m)(max) = 3.9 x 10(6) M(-1) s(-1)] shows a limiting slope of two on the ascending limb indicating the ionization of two essential groups on the free enzyme and/or substrate. One of these groups, with a pK(a) value of 5.4, is reasonably assigned to the 6-carboxylate moiety of 2a (pK(a)(COOH) = 5.4). This assignment is supported by the slope of unity for the ascending limb of log (k(cat)/K-m) versus pH for 2b which lacks this group. Thus a negative charge at the 6-position is important for substrate binding and catalysis. The other group (pK(a2) = 5.2) most likely represents a perturbed pK(a) for the general base Pro-1 (pK(a)(true) = 6.4). The descending limb of log k(cat)/K-m vs pH for 2a has a slope of unity and was fit to a single pK(a3) = 10.3 +/- 0.2. The pH dependence of k(cat) for 2a gives pK(a) values for the ES complex (pK(ES)) of 6.5 and 9.6. On the basis of these results, an isomerization mechanism involving general-base catalysis by a low pK(a) proline-1 and electrophile catalysis by an as yet unidentified enzymic general-acid (pK(a) = 9.0) is proposed.
