Electrostatic effects play a central role in cold adaptation of trypsin.

Organisms that live in constantly cold environments have to adapt their metabolism to low temperatures, but mechanisms of enzymatic adaptation to cold environments are not fully understood. Cold active trypsin catalyses reactions more efficiently and binds ligands more strongly in comparison to warm active trypsin. We have addressed this issue by means of comparative free energy calculations studying the binding of positively charged ligands to two trypsin homologues. Stronger inhibition of the cold active trypsin by benzamidine and positively charged P1-variants of BPTI is caused by rather subtle electrostatic effects. The different affinity of benzamidine originates solely from long range interactions, while the increased binding of P1-Lys and -Arg variants of BPTI is attributed to both long and short range effects that are enhanced in the cold active trypsin compared to the warm active counterpart. Electrostatic interactions thus provide an efficient strategy for cold adaptation of trypsin.