title: Kinetic and structural properties of triosephosphate isomerase from Helicobacter pylori.
abstract: 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.
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, Republic of China.
authors.forename: Chen-Hsi
authors.initials: CH
authors.lastname: Chu
authors.forename: Yi-Ju
authors.initials: YJ
authors.lastname: Lai
authors.forename: Haimei
authors.initials: H
authors.lastname: Huang
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D016480:Helicobacter pylori / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation; D011487:Protein Conformation; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D014305:Triose-Phosphate Isomerase