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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
Characterization of a New Allelic Variant of Triosephosphate Isomerase from the LNCaP Human Prostate Cancer Cell
Valeria Guzmán-Luna1, Leticia Olvera-Rodríguez1, Peniel Bustamante-Villalobos1
1Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, 62250Cuernavaca, Morelos, México.
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 μM and 356 μM 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.
Insights
The G233D variant of triosephosphate isomerase (TIM) shows reduced activity in a key step of glycolysis. This altered enzyme function may enhance the flow of glyceraldehyde-3-phosphate (GAP) through the glycolytic pathway in tumor cells.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- The glycolytic pathway is crucial for tumor cell metabolism.
- Triosephosphate isomerase (TIM) is a key enzyme in glycolysis, catalyzing the interconversion of D-glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).
- A novel G233D TIM variant was identified in prostate cancer cells, necessitating biochemical characterization.
Purpose of the Study:
- To conduct the first biochemical and biophysical characterization of the G233D allelic variant of TIM.
- To compare the enzymatic properties and stability of the G233D variant with the wild-type (WT) TIM.
Main Methods:
- Enzyme kinetics (Michaelis-Menten) were determined for both substrates (GAP and DHAP).
- The effect of the competitive inhibitor phosphoenolpyruvate (PEP) was assessed.
- Thermal stability was analyzed using circular dichroism spectroscopy in the presence and absence of PEP.
Main Results:
- The G233D variant displayed a 4-fold lower catalytic rate (kcat) for GAP to DHAP isomerization compared to WT TIM.
- Inhibition constants (Ki) and IC50 values for the G233D variant were comparable to WT TIM.
- PEP binding induced increased thermal unfolding cooperativity in the G233D variant, indicating a more rigid conformation.
Conclusions:
- The reduced catalytic activity of the G233D TIM variant in DHAP formation may enhance GAP flux through glycolysis.
- This finding has implications for understanding metabolic reprogramming in prostate cancer.
