Schulz Jenny M, Watson Alice L, Sanders Rebecca, Ross Kerry L, Thoden James B, Holden Hazel M, Fridovich-Keil Judith L
Graduate Program in Nutrition and Health Sciences, Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 2004 Jul 30;279(31):32796-803. doi: 10.1074/jbc.M405005200. Epub 2004 Jun 2.
UDP-galactose 4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.
UDP-半乳糖4'-表异构酶（GALE）在Leloir途径的最后一步催化UDP-半乳糖和UDP-葡萄糖相互转化。与大肠杆菌的该酶不同，人源GALE（hGALE）还能高效催化一对更大的底物相互转化：UDP-N-乙酰半乳糖胺和UDP-N-乙酰葡糖胺。这种底物特异性差异的基础一直不明。然而，最近的X射线晶体学数据不仅预测了关键的活性位点残基，还表明活性位点裂隙体积的差异可能是决定GALE底物选择性的关键因素。我们在此报告对这一假设的直接验证。简而言之，我们构建了四个取代等位基因：S132A、Y157F、S132A/Y157F和C307Y-hGALE。虽然前三个取代预计会破坏催化活性，但第四个取代预计会减小活性位点裂隙体积，从而限制较大底物而非较小底物的进入或旋转。所有四个等位基因均在酿酒酵母的无背景菌株中表达，并针对UDP-半乳糖和UDP-N-乙酰半乳糖胺的活性进行了表征。S132A/Y157F和C307Y-hGALE蛋白也在毕赤酵母中过表达并纯化用于分析。在所有测试形式中，Y157F、S132A和Y157F/S132A-hGALE蛋白对两种底物均表现出完全失活。相比之下，C307Y-hGALE对UDP-半乳糖表现出正常活性，但对UDP-N-乙酰半乳糖胺完全失活。这些结果共同验证了野生型hGALE的晶体结构，并充分支持了307位残基作为守门人介导底物进入hGALE活性位点的假设。
