Ternary complex formation and induced asymmetry in orotate phosphoribosyltransferase
Ternary complex formation and induced asymmetry in orotate phosphoribosyltransferase
复制标题
DOI：
10.1021/bi701023z
复制
发表时间：
2007-12-11
期刊：
影响因子：
2.9
通讯作者：
Hurley, Thomas D.
中科院分区：
文献类型：
--
作者：
Gonzalez-Segura, Lilian;Witte, John F.;Hurley, Thomas D.
乳清酸磷酸核糖基转移酶（OPRTase，EC 2.4.2.10）催化乳清酸（OA）与 PRPP（5-α-D-磷酸核糖 1-二磷酸）的 Mg2+ 依赖性缩合，产生二磷酸（PPi）和核苷酸 OMP（乳清苷 5'-单磷酸）。我们已经确定了三种形式的酿酒酵母 OPRTase 的结构，它们代表不同的结构和酶促中间体。结构包括脱辅基酶（分辨率为 2.35 埃）；酶、Mg2+-PRPP 和 OA 的三元复合物（1.74 埃分辨率）；以及酶与 OMP 的二元产物复合物（1.89 埃分辨率）。虽然酿酒酵母OPRTase的整体结构与鼠伤寒沙门氏菌酶的整体结构相似，但通过比较两种脱辅基酶来判断，从脱辅基酶结构到底物和产物复合物的结构，发生了大的构象转变。这些结构的比较表明，OMP 结构中上罩结构域在结合配体上的旋转平均为 19.5 度，而 OA/Mg2+-PRPP 三元复合物中平均为 24.6 度。正如预期的那样，由残基 104-116 组成的保守环在催化循环期间广泛移动并采用单一稳定构象，以便将底物与三元复合物中的本体溶剂隔离。结合在三元复合物中的 OA 和 Mg2+-PRPP 分子定向为 OA 的 NI 原子正确攻击核糖环的 Cl 原子。底物的这种方向与柔性环的定位相结合，提供了 I 型磷酸核糖基转移酶的催化平衡反应复合物的清晰图像。这些结构中存在的结构不对称性，以及在鼠伤寒沙门氏菌酶的最新结构中发现的结构不对称性，与三元复合物中从一个亚基到相对亚基活性位点的柔性环的闭合相结合，与动力学数据一致[McClard, R. W., et al. 2014]。 (2006) Biochemistry 45, 5330-5342]证明了 OPRTase 的诱导非等价性和协同性。
Orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-alpha-D-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5'-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 angstrom resolution); a ternary complex of enzyme,.Mg2+-PRPP, and OA (1.74 angstrom resolution); and the binary product complex of enzyme with OMP (1.89 angstrom resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5 degrees in the OMP structure and an average of 24.6 degrees in the OA/Mg2+-PRPP ternary complex. As expected, the conserved loop, composed of residues 104-116, moves extensively and adopts a single stable conformation during the catalytic cycle in order to sequester the substrates from bulk solvent in the ternary complex. The OA and Mg2+-PRPP molecules bound in the ternary complex are oriented for proper attack of the NI atom of OA onto the Cl atom of the ribose ring. This orientation of substrates, combined with the positioning of the flexible loop, provides a clear picture of a catalytically poised reaction complex for type I phosphoribosyltransferases. The structural asymmetry p resent in these structures, as well as that found in a recent structure of the S. typhimurium enzyme, combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342] that demonstrate induced nonequivalence and cooperativity of OPRTase.