Miller, J. J.; Bahnson, B. J.

Thermolysin, a bacterial zinc metalloprotease, has been previously been reported to exhibit a biphasic kinetic temperature dependence of kcat with a characteristic convex shape. This convex shaping is observed for almost all enzymes which display an Arrhenius break; fumarase is the exception with concave shaping. Here, thermolysin kinetics measured with the tripeptide substrate N-[3-(2-furyl)acryloyl]-Phe-Leu-Ala (FAFLA) resulted in a concave Arrhenius plot, characterized by a 30 kJ/mol increase in enthalpy and entropy of activation, in contrast to the typical 30 kJ/mol decrease. Although the shape of the Arrhenius break differs, ionic strength and macromolecular crowding both attenuate the energetic magnitude of the break point, consistent with prior work. It was hypothesized that a different step of the catalytic cycle of thermolysin was represented by kcat with FAFLA to give rise to this new behavior. A 91% dependence of kcat on viscosity and modest solvent isotope effects, both distinct from previously-characterized substrates, indicated that a physical step was responsible for the observed Arrhenius concavity. Hinge bending conformational changes of thermolysin, monitored using the phosphoramidon inhibitor (a FAFLA mimic), exhibited a fully linear temperature dependence, excluding these large-scale motions as the origin of concavity. It was therefore proposed that release of the N-[3-(2-furyl)acryloyl]-Phe product is likely rate limiting since release was proposed to involve a two-step pathway to free the product coordinated to the catalytic Zn2+ of thermolysin. These findings provide a mechanistic framework for seldom-seen concave break point behavior and insights into the contribution of dynamics of physical processes to catalysis.