Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with .alpha.-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism

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Abstract
The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture. Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley. Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN'', with Ki = 2.9 .times. 10-12 M. Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60). This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme. Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin. In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity. CI2 Met .fwdarw. Lys-59 was found to be a temporary inhibitor of subtilisin BPN'' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN''(Glu .fwdarw. Ser-156). The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.