High Formate Concentrations Research Articles

Effects of temperature and ammonium ions on formyltetrahydrofolate synthetase from Clostridium thermoaceticum

NH 4 + and K + but not Na + increase the thermostability of formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) from the thermophile Clostridium thermoaceticum. When the reaction is measured in the direction of synthesis of 10-formyltetrahydrofolate (forward direction), NH 4 + and K + activate the enzyme at low but not at high concentrations of formate. The apparent K m of formate is decreased 10-fold by the presence of 2 mM NH 4Cl. A small decrease in the apparent K m of ATP and tetrahydrofolate also occurs on addition of NH 4Cl. The V of the reaction is not changed. The reverse reaction is stimulated 4-fold by the addition of 20 mM NH 4Cl. Arrhenius plots show that the activation energy for the synthesis of 10-formyltetrahydrofolate is higher below 40–43 °C than above. Plots of apparent K m values for each of the substrates (for the forward reaction) versus temperature, have breaks at 40–43 °C in the presence or absence of NH 4Cl. The fluorescence (at 325 nm) of the enzyme in the absence of NH 4Cl decreases linearly with increasing temperature; however, in the presence of NH 4Cl, the linearly decreasing fluorescence has a slope change at 40 °C. Absorbance of the enzyme at 295 nm increases linearly with increasing temperature with a slope change at 40 °C both in the presence and absence of NH 4Cl. The kinetic and physical data indicate that the enzyme undergoes a temperature-dependent conformational change at 40–43 °C.