Microscopic rate-constants for substrate binding and acylation in cold-adaptation of trypsin I from Atlantic cod

Temperature imposes limits on where life can thrive and this is evident in the evolution of the basic structural properties of proteins. Cold-adaptation of enzymes is one example, where the catalytic rate constant (kcat) is increased compared with hot-acclimated homologous under identical assay conditions. Trypsin I from Atlantic cod (Gadus morhua) has catalytic efficiency (kcat/Km) for amide hydrolysis that is 17-fold larger than observed for bovine trypsin. Here, the individual rate-constants for association of substrate (k1), dissociation of substrate (k−1), and acylation of the enzyme (k2) have been determined using benzoyl-Arg-p-nitroanilide or benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide as substrates. Rather unexpectedly, by far the largest difference (37-fold increase) was observed in k1, the rate constant for binding of substrate. The cold-adaptation of the dissociation and catalytic steps were not as prominent (increased by 3.7-fold). The length of substrate did have an effect by increasing the reaction rate by 70-fold, and again, the step most affected was the initial binding-step.