Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149.
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.
Berger
E
E
Department of Biochemistry, University of Wisconsin-Madison, 1710 University Avenue, Madison, Wisconsin 53705, USA.
Arabshahi
A
A
Wei
Y
Y
Schilling
J F
JF
Frey
P A
PA
D000409:Alanine / Q000235:genetics; D002384:Catalysis; D003903:Deuterium / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D011487:Protein Conformation; D011522:Protons; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D014534:UDPglucose 4-Epimerase / Q000235:genetics / Q000378:metabolism*
D011522:Protons; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D003903:Deuterium; D014534:UDPglucose 4-Epimerase; C020551:galactose epimerase; D000409:Alanine