REGULATION OF TRYPSIN ACTIVITY BY CU2-BINDING SITE( CHELATION OF THE SUBSTRATE)

Lysine 188 of trypsin was replaced with histidine in order to create a metal chelation site in the substrate binding pocket of this protease , built in a metal binding 'switch,' and to be able to modulate its ac tivity at lower pH. The catalytic properties of wild-type and mutant t rypsin were measured with tetrapeptide substrates containing a nitroan ilide leaving group and whole native protein substrate: beta-casein. T he results obtained reveal that K188H mutation does not affect catalyt ic efficiency, raising only slightly (from 6 to 8) the arginine/lysine preference of the mutant and increasing 1.8- and 1.2-fold the second- order rate constant k(cat)/k(m) for arginine- and lysine-containing su bstrates, respectively, Compared with wild-type trypsin, K188H mutant shows, in the absence of Cu2+, a different catalytic activity pattern as a function of pH. The addition of Cu2+ to trypsin K188H induces a 3 0-100-fold increase in K-m, while k(cat) is scarcely decreased, The hy drolytic activity of this mutant can be fully restored by addition of EDTA, In contrast to a chelating active site, a novel mode of metal-de pendent inhibition activity of trypsin with copper is presented, As su ggested by molecular modelling studies, the substrate binding pocket o f the protease is considerably perturbed by vicinal chelation, More ge nerally, this type of transition metal chelate may present wider possi bilities of trypsin activity and specificity modulation than the previ ously accomplished chelation of a histidine moiety from a catalytic tr iad.