Regulation of trypsin activity by Cu2+ chelation of the substrate binding site.
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 activity at lower pH. The catalytic properties of wild-type and mutant trypsin were measured with tetrapeptide substrates containing a nitroanilide leaving group and whole native protein substrate: beta-casein. The results obtained reveal that K188H mutation does not affect catalytic 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)/Km for arginine- and lysine-containing substrates, 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 30-100-fold increase in Km, while k(cat) is scarcely decreased. The hydrolytic activity of this mutant can be fully restored by addition of EDTA. In contrast to a chelating active site, a novel mode of metal-dependent inhibition activity of trypsin with copper is presented. As suggested by molecular modelling studies, the substrate binding pocket of the protease is considerably perturbed by vicinal chelation. More generally, this type of transition metal chelate may present wider possibilities of trypsin activity and specificity modulation than the previously accomplished chelation of a histidine moiety from a catalytic triad.
Briand
L
L
Institut National de la Recherche Agronomique, LEIMA, Nantes, France.
Chobert
J M
JM
Tauzin
J
J
Declerck
N
N
Léonil
J
J
Mollé
D
D
Tran
V
V
Haertlé
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D002614:Chelating Agents; D003198:Computer Simulation; D003300:Copper / Q000378:metabolism*; D004926:Escherichia coli; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
D002364:Caseins; D002614:Chelating Agents; D006639:Histidine; D003300:Copper; D014357:Trypsin; D008239:Lysine