Macromolecular Crowding Effects on the non-Arrhenius Break Point of Thermolysin

• Mikul Duggal, Biochemistry, University of Delaware

• Brian Bahnson, Department of Chemistry and Biochemistry, University of Delaware
• Jared Miller, Department of Chemistry and Biochemistry, University of Delaware

Previous studies have documented biphasic transitions in the typically linear kinetic temperature dependences of enzymes, as modeled by the Arrhenius equation. These non-Arrhenius break points are classically explained by changes in the rate-limiting step of the reaction, which can potentially be linked to changes in conformational dynamics of a protein  across the break point temperature. Thermolysin, a thermostable bacterial metalloprotease derived from Bacillus thermoproteolyticus, demonstrates one such break point at 26°C. Since the break point temperature lies far outside of the 70°C optimal temperature of thermolysin and previous experiments were performed in dilute buffer solutions that do not adequately reflect in vivo conditions, the characterization of this anomalous behavior in the context of its biological significance warrants consideration. Due to the large-scale open-to-closed conformational change thermolysin undergoes in its catalytic cycle, it was hypothesized that macromolecular crowding, modeled by Ficoll-70, may exert a significant effect on the break point temperature and associated thermodynamic parameters. In the range of 0-15% Ficoll-70, the kinetic temperature dependence of thermolysin reflected successively greater linear character, reaching an apparent loss of biphasic character around 10% Ficoll-70. In investigating the underlying kinetic parameters, both the kcat and Km of succinylcasein showed biphasic behavior with increasing concentrations of Ficoll-70 .