Kinetic Data of D-Glyceraldehyde-3-Phosphate Dehydrogenase from HeLa Cells

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bis-phospho-D-glycerate, and as such participates in the glycolytic conversion of glucose to pyruvic acid in most living organisms. The glycolytic pathway plays an important role in tumor cells, but it is not clear whether the enzyme kinetics of GAPDH or their response to inhibitors, substrates, and cofactors differ between tumor cells and normal cells. To obtain a tumor-derived GAPDH sample, HeLa cells were chosen on the basis of their homogeneous differentiation pattern and ease of harvesting. We carried out experiments to investigate whether the enzyme kinetics of purified GAPDH from HeLa cells were altered in the presence of reagents containing sulfhydryl groups, divalent metal ions, and cellular metabolites such as nucleotides and coenzymes. The kinetic data were compared with data for GAPDH from normal tissue. GAPDH from HeLa cells was activated by 2-mercaptoethanol and dithioerythritol. The maximum activation was obtained at a 1 mM concentration of each reducing agent. Cupric and mercuric ions (1 mM), as well as p-chloro and phydroxymercuribenzoate (10 M), fully inhibited enzymatic activity. Among the nucleotides tested, 3–5-cyclic AMP (cAMP) was the most effective inhibitor at 30 mM concentration, with a relative activity of 22.79 (±1.76), which was significantly different (p 0.05) from that of the control, which had 100% activity in the absence of adenine nucleotide. Enzyme inhibition by adenine nucleotides appeared to be via competition with NAD+. The apparent inhibition constants (Ki) for ADP, 5-AMP, and cAMP were 2.1 mM, 1.0 mM, and 0.6 mM, respectively. GAPDH from HeLa cells was inactivated when incubated in the presence of G3P or NADH at 37 °C, and in both cases the presence of 2-mercaptoethanol protected the enzyme against inhibition. The presence of EDTA did not affect the inactivation of the enzyme by NADH, which suggested that the inactivation of HeLa GAPDH by NADH is not related to the presence of heavy metal ions. Our kinetic analysis showed that although the GAPDH of HeLa cells has a lower specific activity and stability compared with GAPDH from normal tissue, its kinetic characteristics were similar, reinforcing the key role of this enzyme in the metabolism of tumor cells. Keywords: Adenine nucleotides, D-Glyceraldehyde-3-phosphate dehydrogenase, GAPDH, HeLa cells, p-hydroxymecuribenzoate, sulfhydryl reagents.