C-13 NMR analysis of electrostatic interactions between NAD(+) and active site residues of UDP-galactose 4-epimerase: Implications for the activationinduced by uridine nucleotidest

UDP-galactose 4-epimerase contains the coenzyme NAD(+) bound tightly at the active site. NAD(+) functions as the coenzyme for the interconversion of U DP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. The epimerase structur e and spectrophotometric data indicate that NAD(+) may engage in electrosta tic interactions with amino acid side chains that may regulate the reactivi ty of NAD(+). In this work, we carried out NMR studies of [nicotinamide-4-C -13]NAD(+) bound to wild-type epimerase and epimerases mutated at amino aci d residues in contact with NAD(+). The 4-C-13 NMR chemical shifts revealed the following: The 4-C-13 chemical shift in wild-type epimerase is 149.9 pp m; mutation of Ser 124 to Ala changes it slightly by 0.2 ppm to 150.1 ppm; mutation of Tyr 149 to Phe results in a downfield perturbation of 2.7 ppm t o 152.6 ppm; and the simultaneous mutation of Ser 124 to Ala and Tyr 149 to Phe also causes a downfield perturbation of 2.8 ppm to 152.7 ppm. Mutation of Lys 1.53 to Met results in a C-13 chemical shift of 150.8 ppm, which is 0.9 ppm downfield from that of wild type and 1.8 ppm upfield from that of Y149F-epimerase. The 13C chemical shifts of nicotinamide C4 of NAD(+) in th ese epimerases are correlated with their respective reactivities with NaBH3 CN. In addition, reactivity of NAD(+) in wild-type and S124A-epimerases dis plays pH dependence, with higher rates at lower pH where Tyr 149 in these t wo enzymes is protonated. The results support an electrostatic model in whi ch repulsion between positively charged Lys 153 and N1 of the nicotinamide ring increases the reactivity of NAD(+), while the phenolate of Tyr 149 opp oses the positive electrostatic field and attenuates the reactivity of NAD( +). Ser 124 has very little effect on the electron distribution within the nicotinamide ring or the reactivity of NAD(+). The effects of binding the s ubstrate analogue P-1-uridyl-P-2-methyl diphosphate (Me-UDP) on the 4-13C c hemical shifts are opposite to those induced by the mutations. MeUDP pertur bs the 4-13C chemical shift 2.9 ppm downfield in the wild-type and S124A-ep imerases but has little or no effect in the cases of Y149F- or K153M-epimer ases. The results support the postulate that NAD+ activation induced by uri dine nucleotides is brought about by a conformational change of epimerase t hat repositions Tyr 149 at an increased distance from nicotinamide N1 of NA D(+) while maintaining the electrostatic repulsion between Lys 153 and nico tinamide N1 of NAD(+).