EFFECTS OF SERPIN BINDING ON THE TARGET PROTEINASE - GLOBAL STABILIZATION, LOCALIZED INCREASED STRUCTURAL FLEXIBILITY, AND CONSERVED HYDROGEN-BONDING AT THE ACTIVE-SITE

The binding of human alpha(1)-proteinase inhibitor to rat trypsin was shown by NMR spectroscopy to raise the pK(a)' of His(57) in the active site but not to disrupt the hydrogen bond between His(57) and Asp(102 ) Similar NMR results were observed for the Asp(189) to serine mutant of rat trypsin, which is much more stable than wild-type trypsin again st autoproteolysis as the result of mutation of the residue at the bas e of the specificity pocket, This mutant was used in further studies a imed at determining the extent of the conformational transition in try psin that accompanies serpin binding and leads to disruption of the ca talytic activity of the proteinase such that the inhibitor complex is trapped at the acyl enzyme intermediate stage. The stability of rat tr ypsin toward thermal denaturation was found to be lower in the free en zyme than in the complex with alpha(1)-proteinase inhibitor. This sugg ests that the complex contains extensive protein-protein interactions that stabilize overall folding. On the other hand, previous investigat ions have shown that the proteinase in serpin-proteinase complexes bec omes more susceptible to limited proteolysis, suggesting that the conf ormational change that accompanies binding leads to the exposure of su sceptible loops in the enzyme. The existence of this type of conformat ional change upon complex formation has been confirmed here by investi gation of the rate of cleavage of disulfide linkages by added dithioth reitol. This study revealed that, despite the increased stability of t rypsin in the complex, one or more of its disulfide bridges becomes mu ch more easily reduced. We suggest that the process of complex formati on with alpha(1)-proteinase inhibitor converts trypsin D189S into an i nactive, loose structure, which serves as a ''conformational trap'' of the enzyme that prevents catalytic deacylation. It is also proposed t hat plastic region(s) of the activation domain of trypsin may play a c rucial role in this inhibitor-induced structural rearrangement.