THE EFFECTS OF TEMPERATURE ON THE KINETICS AND STABILITY OF MESOPHILIC AND THERMOPHILIC 3-PHOSPHOGLYCERATE KINASES

The effects of temperature on the kinetic parameters k(cat) and K-m, f or three isolates of the highly conserved monomeric enzyme 3-phosphogl ycerate kinase (PGK), were investigated in detail using a rapid automa ted kinetics apparatus. PGK was purified from the thermophilic bacteri um Thermoanaerobacter sp. Rt8.G4 (optimum growth temperature 68 degree s C), the mesophile Zymomonas mobilis (optimum growth temperature 32 d egrees C) and a second, unidentified, soil mesophile designated unid A (optimum growth temperature 27 degrees C). The kinetic behaviour with temperature of each PGK preparation was distinct, despite the conserv ed nature of the enzyme. The k(cat) values increased with temperature, but not as rapidly exponentially, as might be expected from the Arrhe nius equation, Maximum k(cat) values were at much higher temperatures than the optimum growth temperatures for the mesophiles, but for the t hermophile the temperature of maximum k(cat) was close to its optimum growth temperature. K-m values were in general nearly constant through the lower temperature ranges, but increased substantially as the opti mum temperature (highest k(cat)) was passed. Thermal irreversible dena turation of the PGK proteins was also investigated by measuring loss o f activity over time. In a dilute buffer, Arrhenius plots for denatura tion were linear, and the calculated apparent energy of activation (E- act) for denaturation for the thermophilic PGK was 600 kJ.mol(-1), whe reas for the mesophilic enzymes the values were 200-250 kJ.mol(-1). In the presence of substrates, a considerable stabilization occurred, an d in the case of the Z. mobilis enzyme, the apparent E-act was increas ed to 480 kJ.mol(-1). A theoretical explanation for these observations is presented. Comparing the kinetics data with irreversible denaturat ion rates determined at relevant temperatures, it was clear that k(cat ) values reached a maximum, and then decreased with higher temperature before irreversible denaturation had any significant influence.