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 (k(cat)) is increased compared with hot-acclimated homologous under identical assay conditions. Trypsin I from Atlantic cod (Gadus morhua) has catalytic efficiency (k(cat)/K(m)) for amide hydrolysis that is 17-fold larger than observed for bovine trypsin. Here, the individual rate-constants for association of substrate (k(1)), dissociation of substrate (k(-1)), and acylation of the enzyme (k(2)) 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 k(1), 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.
Asgeirsson
Bjarni
B
Department of Biochemistry, Science Institute, University of Iceland, Dunhaga 3, IS-107 Reykjavik, Iceland. bjarni@raunvis.hi.is
Cekan
Pavol
P
D000215:Acylation; D000222:Adaptation, Physiological; D000818:Animals; D003080:Cold Temperature; D005399:Fishes / Q000502:physiology*; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism / Q000502:physiology*
D014357:Trypsin