Abstract A growing number of enzymes demonstrate deviations from typical linear kinetic temperature dependencies as modeled by the Arrhenius and Eyring equations. These deviations may manifest as biphasic temperature dependencies consisting of two individually linear regions meeting at a break point, which suggests a change of conformation or dynamics at this critical temperature. Thermolysin is a thermostable metalloprotease produced by Bacillus thermoproteolyticus with previously characterized break point behavior at 26°C. Since this break point was examined in dilute buffer solutions and occurs at temperatures nearly 50°C lower than the optimal temperature of the enzyme, one must consider that the break point may be the result of additional degrees of conformational flexibility permitted by less complex in vitro conditions. To assess the biological significance of this Arrhenius break, succinylated bovine β‐casein was used as the substrate in a 2,4,6‐trinitrobenzenesulfonic acid‐linked kinetic assay with elevated concentrations of ionic strength, viscosity, or the macromolecular crowding agent Ficoll‐70. It was determined that changes to the K M for succinylcasein did not significantly contribute to the break point behavior, with a change in k cat behavior being responsible for the −28 kJ/mol difference in enthalpy and entropy of activation across the break point. With the addition of 1 M NaCl or 15% (w/w) Ficoll‐70, this energetic difference decreased to −8.4 or −11 kJ/mol, respectively, with Ficoll‐70 effectively linearizing the full Arrhenius plot. Both of these effects suggest a decreased severity of the break point in complex environments which may mirror behavior of thermolysin in vivo.
Miller et al. (Mon,) studied this question.