Chu Chen-Hsi, Lai Yi-Ju, Huang Haimei, Sun Yuh-Ju
Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, Republic of China.
Proteins. 2008 Apr;71(1):396-406. doi: 10.1002/prot.21709.
Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate in the glycolysis-gluconeogenesis metabolism pathway. The Helicobacter pylori TIM gene (HpTIM) was cloned, and HpTIM was expressed and purified. The enzymatic activity of HpTIM for the substrate GAP was determined (K(m) = 3.46 +/- 0.23 mM and k(cat) = 8.8 x 10(4) min(-1)). The crystal structure of HpTIM was determined by molecular replacement at 2.3 A resolution. The overall structure of HpTIM was (beta/alpha)beta(beta/alpha)(6), which resembles the common TIM barrel fold, (beta/alpha)(8); however, a helix is missing after the second beta-strand. The conformation of loop 6 and binding of phosphate ion suggest that the determined structure of HpTIM was in the "closed" state. A highly conserved Arg-Asp salt bridge in the "DX(D/N)G" motif of most TIMs is absent in HpTIM because the sequence of this motif is "(211)SVDG(214)." To determine the significance of this salt bridge to HpTIM, four mutants, including K183S, K183A, D213Q, and D213A, were constructed and characterized. The results suggest that this conserved salt bridge is not essential for the enzymatic activity of HpTIM; however, it might contribute to the conformational stability of HpTIM.
磷酸丙糖异构酶（TIM）在糖酵解-糖异生代谢途径中催化磷酸二羟丙酮和D-甘油醛-3-磷酸之间的相互转化。克隆了幽门螺杆菌TIM基因（HpTIM），并对HpTIM进行了表达和纯化。测定了HpTIM对底物甘油醛-3-磷酸（GAP）的酶活性（米氏常数K(m)=3.46±0.23 mM，催化常数k(cat)=8.8×10⁴ min⁻¹）。通过分子置换法在2.3 Å分辨率下测定了HpTIM的晶体结构。HpTIM的整体结构为（β/α）β（β/α）⁶，类似于常见的TIM桶状折叠结构（β/α）⁸；然而，在第二条β链之后缺少一个螺旋。环6的构象和磷酸根离子的结合表明所测定的HpTIM结构处于“关闭”状态。大多数TIMs的“DX(D/N)G”基序中高度保守的精氨酸-天冬氨酸盐桥在HpTIM中不存在，因为该基序的序列为“(211)SVDG(214)”。为了确定该盐桥对HpTIM的重要性，构建并表征了四个突变体，包括K183S、K183A、D213Q和D213A。结果表明，这个保守的盐桥对HpTIM的酶活性不是必需的；然而，它可能有助于HpTIM的构象稳定性。
