Acid−Base Catalysis by UDP-Galactose 4-Epimerase: Correlations of Kinetically Measured Acid Dissociation Constants with Thermodynamic Values for Tyrosine 149

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Abstract
The steady-state kinetic parameters for epimerization of UDP-galactose by UDP-galactose 4-epimerase from Escherichia coli (GalE), Y149F-GalE, and S124A-GalE have been measured as a function of pH. The deuterium kinetic isotope effects for epimerization of UDP-galactose-C-d7 by these enzymes have also been measured. The results show that the activity of wild-type GalE is pH-independent in the pH range of 5.5−9.3, and there is no significant deuterium kinetic isotope effect in the reaction of UDP-galactose-C-d7. It is concluded that the rate-limiting step for epimerization by wild-type GalE is not hydride transfer and must be either a diffusional process or a conformational change. Epimerization of UDP-galactose-C-d7 by Y149F-GalE proceeds with a pH-dependent deuterium kinetic isotope effect on kcat of 2.2 ± 0.4 at pH 6.2 and 1.1 ± 0.5 at pH 8.3. Moreover, the plot of log kcat/Km breaks downward on the acid side with a fitted value of 7.1 for the pKa. It is concluded that the break in the pH−rate profile arises from a change in the rate-limiting step from hydride transfer at low pH to a conformational change at high pH. Epimerization of UDP-galactose-C-d7 by S124A-GalE proceeds with a pH-independent deuterium kinetic isotope effect on kcat of 2.0 ± 0.2 between pH 6 and 9. Both plots of log kcat and log kcat/Km display pH dependence. The plot of log kcat versus pH breaks downward with a pKa of 6.35 ± 0.10. The plot of log kcat/Km versus pH is bell-shaped, with fitted pKa values of 6.76 ± 0.09 and 9.32 ± 0.21. It is concluded that hydride transfer is rate-limiting, and the pKa of 6.7 for free S124A-GalE is assigned to Tyr 149, which displays the same value of pKa when measured spectrophotometrically in this variant. Acid−base catalysis by Y149F-GalE is attributed to Ser 124, which is postulated to rescue catalysis of proton transfer in the absence of Tyr 149. The kinetic pKa of 7.1 for free Y149F-GalE is lower than that expected for Ser 124, as proven by the pH-dependent kinetic isotope effect. Epimerization by the doubly mutated Y149F/S124A-GalE proceeds at a kcat that is lower by a factor of 107 than that of wild-type GalE. This low rate is attributed to the synergistic actions of Tyr 149 and Ser 124 in wild-type GalE and to the absence of any internal catalysis of hydride transfer in the doubly mutated enzyme.