Thoden J B, Ruzicka F J, Frey P A, Rayment I, Holden H M
Department of Biochemistry, University of Wisconsin-Madison, 53705, USA.
Biochemistry. 1997 Feb 11;36(6):1212-22. doi: 10.1021/bi9626517.
Galactose-1-phosphate uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5'-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 A resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 68 A, b = 58 A, c = 189 A, and beta = 100 degrees and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues critically involved in sugar binding include Asn 153 and Gly 159 from the first subunit and Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, and Gln 323 from the second subunit. The uridylyltransferase is able to accommodate both UDP-galactose and UDP-glucose substrates by simple movements of the side chains of Glu 317 and Gln 323 and by a change in the backbone dihedral angles of Val 314. The removal of the imidazole group at position 166 results in little structural perturbation of the polypeptide chain backbone when compared to the previously determined structure for the wild-type enzyme. Instead, the cavity created by the mutation is partially compensated for by the presence of a potassium ion and its accompanying coordination sphere. As such, the mutant protein structures presented here represent valid models for understanding substrate recognition and binding in the native galactose-1-phosphate uridylyltransferase.
1-磷酸半乳糖尿苷酰转移酶在半乳糖代谢中起着关键作用，它催化尿苷5'-磷酸基团从UDP-葡萄糖转移至1-磷酸半乳糖。大肠杆菌中的该酶由两个相同的亚基组成。已通过单晶X射线衍射分析确定并精修了该酶/UDP-葡萄糖和UDP-半乳糖复合物的结构，其中催化亲核试剂组氨酸166已被甘氨酸残基取代，分辨率达到1.8埃。研究中使用的晶体属于空间群P2(1)，晶胞参数为a = 68埃，b = 58埃，c = 189埃，β = 100°，不对称单位中有两个二聚体。这些酶/底物复合物的模型表明，尿苷酰转移酶的活性位点由二聚体中两个亚基贡献的氨基酸残基形成。那些与糖结合至关重要的氨基酸残基包括来自第一个亚基的天冬酰胺153和甘氨酸159，以及来自第二个亚基的赖氨酸311、苯丙氨酸312、缬氨酸314、酪氨酸316、谷氨酸317和谷氨酰胺323。尿苷酰转移酶能够通过谷氨酸317和谷氨酰胺323侧链的简单移动以及缬氨酸314主链二面角的变化来容纳UDP-半乳糖和UDP-葡萄糖底物。与先前确定的野生型酶结构相比，166位咪唑基团的去除对多肽链主链几乎没有结构扰动。相反，突变产生的空腔部分地由钾离子及其伴随的配位球所补偿。因此，这里呈现的突变蛋白结构是理解天然1-磷酸半乳糖尿苷酰转移酶中底物识别和结合的有效模型。
