13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides.
13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides.
复制标题
NAD 和 UDP-半乳糖 4-差向异构酶活性位点残基之间的静电相互作用的 13C NMR 分析：对尿苷核苷酸诱导的激活的影响。
作者：
Wei,Y;Lin,J;Frey,PA
udp -半乳糖4- epimase在活性位点含有紧密结合的辅酶NAD+。NAD+作为辅酶，通过可逆地介导udp -半乳糖和udp -葡萄糖脱氢成常见的中间体udp -4-酮己基吡喃苷，从而实现udp -半乳糖和udp -葡萄糖相互转化。epimase结构和分光光度数据表明，NAD+可能与氨基酸侧链发生静电相互作用，从而调节NAD+的反应性。在这项工作中，我们对[烟酰胺-4- 13c]NAD+结合野生型外链酶和与NAD+接触的氨基酸残基突变的外链酶进行了核磁共振研究。4-13C核磁共振化学位移表明：野生型epimase的4-13C化学位移为149.9 ppm；Ser 124对Ala的突变使其略微改变0.2 ppm至150.1 ppm；tyr149对Phe的突变导致2.7 ppm至152.6 ppm的下场扰动；Ser 124突变为Ala和tyr149突变为Phe也会引起2.8 ppm至152.7 ppm的下场扰动。Lys 153向Met的突变导致a13C的化学位移为150.8 ppm，比野生型低0.9 ppm，比y149f - epimase高1.8 ppm。NAD+的烟酰胺C4的13c化学位移与它们各自与NaBH3CN的反应性相关。此外，NAD+在野生型和s124a -外膜酶中的反应性表现出pH依赖性，在较低的pH下，这两种酶中的tyr149被质子化，反应率较高。结果表明，带正电的Lys 153与烟酰胺环N1之间的斥力增强了NAD+的反应性，而Tyr 149的酚酸盐与带正电的静电场相反，减弱了NAD+的反应性。Ser 124对烟酰胺环内的电子分布和NAD+的反应性影响很小。结合底物类似物ep1 -尿苷基- p2 -二磷酸甲酯（Me-UDP）对4-13C化学位移的影响与突变诱导的相反。MeUDP对野生型和s124a -外膜酶的4-13C化学位移向下影响2.9 ppm，但对Y149F-或k153m -外膜酶的影响很小或没有影响。结果支持了尿苷核苷酸诱导NAD+激活的假设，即epimase的构象变化使Tyr 149与NAD+的烟酰胺N1的距离增加，同时保持了Lys 153与NAD+的烟酰胺N1之间的静电斥力。
UDP-galactose 4-epimerase contains the coenzyme NAD+bound tightly at the active site. NAD+functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. The epimerase structure and spectrophotometric data indicate that NAD+may engage in electrostatic interactions with amino acid side chains that may regulate the reactivity of NAD+. In this work, we carried out NMR studies of [nicotinamide-4-13C]NAD+bound to wild-type epimerase and epimerases mutated at amino acid residues in contact with NAD+. The 4-13C NMR chemical shifts revealed the following: The 4-13C chemical shift in wild-type epimerase is 149.9 ppm; 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 to 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 153 to Met results in a13C 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. The13C chemical shifts of nicotinamide C4 of NAD+in these epimerases are correlated with their respective reactivities with NaBH3CN. In addition, reactivity of NAD+in wild-type and S124A-epimerases displays pH dependence, with higher rates at lower pH where Tyr 149 in these two enzymes is protonated. The results support an electrostatic model in which repulsion between positively charged Lys 153 and N1 of the nicotinamide ring increases the reactivity of NAD+, while the phenolate of Tyr 149 opposes 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 substrate analogueP1-uridyl-P2-methyl diphosphate (Me-UDP) on the 4-13C chemical shifts are opposite to those induced by the mutations. MeUDP perturbs the 4-13C chemical shift 2.9 ppm downfield in the wild-type and S124A-epimerases but has little or no effect in the cases of Y149F- or K153M-epimerases. The results support the postulate that NAD+activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr 149 at an increased distance from nicotinamide N1 of NAD+while maintaining the electrostatic repulsion between Lys 153 and nicotinamide N1 of NAD+.