MECHANISMS OF TEMPORARY INHIBITION IN STREPTOMYCES SUBTILISIN INHIBITOR INDUCED BY AN AMINO-ACID SUBSTITUTION, TRYPTOPHAN-86 REPLACED BY HISTIDINE

Just one amino acid substitution (Trp86 replaced by His), which is more than 30 angstrom away from the reactive site, changed the inhibitor, Streptomyces subtilisin inhibitor (SSI), into a temporary inhibitor without a change in the inhibition constant. When the inhibitor was in excess of subtilisin BPN', the wild-type SSI was stable under protease attack, while the mutant inhibitor was hydrolyzed to peptide fragments in an all-or-none manner. The mechanism of this temporary inhibition induced by the amino acid substitution was studied on the basis of structural, thermodynamic, and kinetic data obtained by a combined use of NMR, hydrogen-deuterium exchange, differential scanning calorimetry, and gel filtration HPLC. The mutation did not induce major structural changes, and in particular, the structure of the enzyme-binding region was virtually unaffected. The denaturation temperature of SSI, however, was decreased by 10 deg upon mutation, although it still remained a thermostable protein with a denaturation temperature of 73-degrees-C. Furthermore, the activation enthalpy for denaturation was reduced dramatically, to half that of the wild type. When the mutated SSI is present in excess of the enzyme, the proteolysis followed first-order reaction kinetics with respect to the total concentration of the mutated SSI molecules present. From these combined results, we conclude that the proteolysis proceeds not through the native form of the inhibitor in the inhibitor-enzyme complex but through the denatured (unfolded) form of the inhibitor whose fraction is increased by the mutation. This conclusion states that the necessary condition for being a serine protease inhibitor lies not only in the design of the reactive site structure that is highly resistant to protease attack but also in the suppression of such structural fluctuation that brings about cooperative denaturation. In contrast, when the protease existed in excess of the mutated inhibitor, the proteolysis reaction was accelerated by more than 2 orders of magnitude. Furthermore, the reaction occurred even in the wild-type SSI at a comparable rate as in the mutated protein. This indicates that in the enzyme excess case another, more efficient digestion mechanism involving fluctuation within the native manifold of the inhibitor dominates.