Kinetic studies on acid denaturation and renaturation of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.
Fast reversible conformational changes induced by down and up pH-jumps of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor (abbreviated to SSI), was studied kinetically with the stopped-flow method by monitoring the change in tryptophan fluorescence. Both the acid denaturation and the renaturation proceeded in two phases. The pH-dependence of the apparent first-order rate constants and of the magnitudes of the fluorescence changes examined in the range of pH 1.7-7.0 were essentially consistent with a two-step sequential mechanism involving a common intermediate for denaturation and renaturation with rapid protonation equilibria, as follows: (Formula: see text) where N, N', and D denote the native, the intermediate, and the denatured states of the SSI molecule, respectively. The parameters were evaluated as follows: pK1 = 6.0, pK2 = 4.8, pK3 = pK4 = 2.5, k1 = 1.0 s-1, k2 = 0.13 s-1, k3 = 0.05 s-1, and k4 = 60 s-1 (at 25 degrees C). The contributions of Step I and Step II to the total fluorescence change were estimated to be 20.5% and 79.5%, respectively. It is noteworthy that the overall renaturation is faster than the denaturation. The rate constant of restoration of binding ability of SSI to alpha-chymotrypsin (43 s-1), measured by using proflavine as indicator, was of the same order of magnitude as that of the faster step of the renaturation (k4 = 60 s-1), which indicated that the inhibitory activity was lost in the N' leads to D process in Step II.
D001426:Bacterial Proteins / Q000378:metabolism*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins / Q000037:antagonists & inhibitors*
D001426:Bacterial Proteins; C018150:subtilisin inhibitor protein, Streptomyces; D013381:Subtilisins