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

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-d(7) 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-d(7). 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-d(7) by Y149F-GalE proceeds with a pH-dependent deuterium kinetic isotope effect on k(cat) of 2.2 +/- 0.4 at pH 6.2 and 1.1 +/- 0.5 at pH 8.3. Moreover, the plot of log k(cat)/K(m) breaks downward on the acid side with a fitted value of 7.1 for the pK(a). 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-d(7) by S124A-GalE proceeds with a pH-independent deuterium kinetic isotope effect on k(cat) of 2.0 +/- 0.2 between pH 6 and 9. Both plots of log k(cat) and log k(cat)/K(m) display pH dependence. The plot of log k(cat) versus pH breaks downward with a pK(a) of 6.35 +/- 0.10. The plot of log k(cat)/K(m) versus pH is bell-shaped, with fitted pK(a) values of 6.76 +/- 0.09 and 9.32 +/- 0.21. It is concluded that hydride transfer is rate-limiting, and the pK(a) of 6.7 for free S124A-GalE is assigned to Tyr 149, which displays the same value of pK(a) 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 pK(a) 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 k(cat) that is lower by a factor of 10(7) 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.

Functional analysis of disease‐causing mutations in human UDP‐galactose 4‐epimerase

“…2). The kinetic parameters determined from these data (Table 1) are similar to those published for human GALE and for GALEs from other species [21,27,29,30]. There is no evidence that human GALE is glycosylated in vivo, and there is no anomalous migration of recombinant human GALE produced in yeast [27], and thus the observed activity probably reflects that of the native enzyme.…”

UDP-hexose 4-epimerases: a view on structure, mechanism and substrate specificity

“…35 Further analysis of the available eGalE structures and spectrophotometric studies revealed a pHdependent charge transfer complex between Tyr149 and NAD. 44 Kinetic experiments with E. coli GalE and its S124A variant confirmed that tyrosine is the base catalyst for hydride transfer. 43,44 In the next step of the mechanism, the transient keto-sugar is retained in the active site by anchoring of the substrate by its UDPgroup while the 4 0 -ketopyranose intermediate undergoes a rotation in the active site.…”

“…44 Kinetic experiments with E. coli GalE and its S124A variant confirmed that tyrosine is the base catalyst for hydride transfer. 43,44 In the next step of the mechanism, the transient keto-sugar is retained in the active site by anchoring of the substrate by its UDPgroup while the 4 0 -ketopyranose intermediate undergoes a rotation in the active site. Finally, the reverse of the first step occurs, namely the transfer of the hydride back to the C4 of the sugar.…”

The structural and molecular biology of type III galactosemia

“…This movement means that the re-reduction reaction is not stereospecific and thus the products are a mix of C-4 isomers (19,20). The redox reaction is facilitated by a tyrosine residue (Tyr-157 in the human enzyme) acting as an active site base (13,21). The bacterial enzyme is highly specific for UDPgalactose, whereas the human enzyme can also catalyse the interconversion of UDP-N-acetylgalactosamine and UDP-Nacetylglucosamine.…”