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 (kcat/Km) and of log kcat 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, pKa values of 6.2 ± 0.3 and 9.0 ± 0.3 for the free enzyme (pKE) and 7.7 ± 0.3 and 8.5 ± 0.3 for the ES complex (pKES) were obtained. The pKE of 6.2 ± 0.3 for 2b represents a true pKa 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, 15N NMR titration of the uniformly 15N-labeled enzyme showed that the pKa of the amino group of Pro-1 is 6.4 ± 0.2, in reasonable agreement with those found by the effect of pH on kcat/Km for 2b (6.2 ± 0.3) and on kinact/KI for 3-BP (6.7 ± 0.3), but three units lower than the pKa of the model compound proline amide (pKa = 9.4 ± 0.2). The pKa values for the two histidine residues of 4-OT, which were measured by 1H NMR (His-6, pKa ≤ 5; His-49, pKa = 5.2 ± 0.2), are at least one pK unit lower than the pKE, excluding these residues as candidates for the general base. A plot of log (kcat/Km) vs pH for the 104-fold more reactive, but sticky substrate 2a [(kcat/Km)max = 3.9 × 106 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 pKa value of 5.4, is reasonably assigned to the 6-carboxylate moiety of 2a (pKaCOOH = 5.4). This assignment is supported by the slope of unity for the ascending limb of log (kcat/ Km) 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 (pKa2 = 5.2) most likely represents a perturbed pKa for the general base Pro-1 (pKatrue = 6.4). The descending limb of log kcat/Km vs pH for 2a has a slope of unity and was fit to a single pKa3 = 10.3 ± 0.2. The pH dependence of kcat for 2a gives pKa values for the ES complex (pKES) of 6.5 and 9.6. On the basis of these results, an isomerization mechanism involving general-base catalysis by a low pKa proline-1 and electrophilic catalysis by an as yet unidentified enzymic general-acid (pKa = 9.0) is proposed.
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