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Kinetic and structural properties of triosephosphate isomerase from Helicobacter pylori
Author(s) -
Chu ChenHsi,
Lai YiJu,
Huang Haimei,
Sun YuhJu
Publication year - 2008
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21709
Subject(s) - triosephosphate isomerase , helicobacter pylori , isomerase , chemistry , kinetic energy , microbiology and biotechnology , physics , biology , biochemistry , enzyme , genetics , quantum mechanics
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 × 10 4 min −1 ). The crystal structure of HpTIM was determined by molecular replacement at 2.3 Å resolution. The overall structure of HpTIM was (β/α)β(β/α) 6 , which resembles the common TIM barrel fold, (β/α) 8 ; however, a helix is missing after the second β‐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. Proteins 2008. © 2007 Wiley‐Liss, Inc.