Dataset Information

Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

ABSTRACT: Background:The glycolytic pathway plays an important role in tumor cells. Triosephosphate isomerase (TIM) catalyzes the reversible isomerization of D-glyceraldehyde-3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) in the glycolysis. Proteomics of a human prostate adenocarcinoma cell line revealed the presence of the G233D TIM variant, a new allelic type whose biochemical properties have not been reported [1]. Objective:Provide the first biochemical and biophysical characterization of the allelic variant G233D of TIM. Methods:The Michaelis-Menten curves using both substrates of TIM were obtained. Also the effect of the competitive inhibitor phosphoenolpyruvate (PEP) was assessed in presence of GAP and DHAP. The thermal stability in absence and presence of PEP was analyzed by circular dichroism spectroscopy. For comparison purposes, all the measurements were carried out on the wild type TIM and variant G233D. Results:The G233D variant exhibited a kcat value 4-fold lower than that of the WT enzyme in the GAP isomerization to DHAP, which is the reverse reaction of the glycolytic pathway. The G233D variant exhibited Ki and IC50 values of 120 ?M and 356 ?M in the presence of several concentrations of GAP and 0.3 mM DHAP, respectively. These inhibition parameters are similar to those exhibited by the WT enzyme. The thermal unfolding cooperativity of G233D variant was significantly increased upon PEP binding, suggesting that the ligand-bound enzyme was trapped in a rigid conformation. Conclusion:We suggest that the flow of GAP through glycolysis could be enhanced by the decreased activity of the G233D variant in the formation of DHAP.

SUBMITTER: Guzman-Luna V

PROVIDER: S-EPMC5652080 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

Guzmán-Luna Valeria V Olvera-Rodríguez Leticia L Bustamante-Villalobos Peniel P Saab-Rincón Gloria G

Current enzyme inhibition 20150801 3

<h4>Background</h4>The glycolytic pathway plays an important role in tumor cells. Triosephosphate isomerase (TIM) catalyzes the reversible isomerization of D-glyceraldehyde-3-phosphate (GAP) to dihydroxyacetone phosphate (DHAP) in the glycolysis. Proteomics of a human prostate adenocarcinoma cell line revealed the presence of the G233D TIM variant, a new allelic type whose biochemical properties have not been reported [1].<h4>Objective</h4>Provide the first biochemical and biophysical characteri ...[more]

PMID: 29123462

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Project description:Two mutations of the phosphodianion gripper loop in chicken muscle triosephosphate isomerase (cTIM) were examined: (1) the loop deletion mutant (LDM) formed by removal of residues 170-173 [Pompliano, D. L., et al. (1990) Biochemistry 29, 3186-3194] and (2) the loop 6 replacement mutant (L6RM), in which the N-terminal hinge sequence of TIM from eukaryotes, 166-PXW-168 (X = L or V), is replaced by the sequence from archaea, 166-PPE-168. The X-ray crystal structure of the L6RM shows a large displacement of the side chain of E168 from that for W168 in wild-type cTIM. Solution nuclear magnetic resonance data show that the L6RM results in significant chemical shift changes in loop 6 and surrounding regions, and that the binding of glycerol 3-phosphate (G3P) results in chemical shift changes for nuclei at the active site of the L6RM that are smaller than those of wild-type cTIM. Interactions with loop 6 of the L6RM stabilize the enediolate intermediate toward the elimination reaction catalyzed by the LDM. The LDM and L6RM result in 800000- and 23000-fold decreases, respectively, in kcat/Km for isomerization of GAP. Saturation of the LDM, but not the L6RM, by substrate and inhibitor phosphoglycolate is detected by steady-state kinetic analyses. We propose, on the basis of a comparison of X-ray crystal structures for wild-type TIM and the L6RM, that ligands bind weakly to the L6RM because a large fraction of the ligand binding energy is utilized to overcome destabilizing electrostatic interactions between the side chains of E168 and E129 that are predicted to develop in the loop-closed enzyme. Similar normalized yields of DHAP, d-DHAP, and d-GAP are formed in LDM- and L6RM-catalyzed reactions of GAP in D2O. The smaller normalized 12-13% yield of DHAP and d-DHAP observed for the mutant cTIM-catalyzed reactions compared with the 79% yield of these products for wild-type cTIM suggests that these mutations impair the transfer of a proton from O-2 to O-1 at the initial enediolate phosphate intermediate. No products are detected for the LDM-catalyzed isomerization reactions in D2O of [1-(13)C]GA and HPi, but the L6RM-catalyzed reaction in the presence of 0.020 M dianion gives a 2% yield of the isomerization product [2-(13)C,2-(2)H]GA.

Dataset Information

Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

Publications

Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell Line: Enzyme Inhibition and Spectroscopic Studies.

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