BACKBONE DYNAMICS OF CHYMOTRYPSIN INHIBITOR .2. EFFECT OF BREAKING THE ACTIVE-SITE BOND AND ITS IMPLICATIONS FOR THE MECHANISM OF INHIBITION OF SERINE PROTEASES

The backbone dynamics of uniformly N-15-labeled chymotrypsin inhibitor 2 (CI2) and of the complex formed by the association of two fragments consisting of residues 20-59 and 60-83 have been studied. A data set consisting of N-15 longitudinal (T-1) and transverse (T-1 rho) relaxat ion times and {H-1}-N-15 NOE enhancements has been measured for all ba ckbone NH groups in both proteins. Information on internal motions has been extracted from these data using the model-free approach to deter mine order parameters (S-2) and effective internal correlation times ( tau(e)). The data indicate that most of the backbone of CI2 is highly constrained (S-2 approximate to 0.9) with the exception of residues in the binding loop (residues 54-64), which have slightly lower order pa rameters. Most of the residues in the CI2(20-59).(60-83) complex are a lso highly constrained (S-2 approximate to 0.9). However, the loss of the covalent bond between Met59 and Glu60 leads to a large increase in the mobility of residues in the loop region. The residues in the firs t half of the loop region have significantly lower order parameters th an those in the second half of the loop. This observation suggests tha t the NH2 group that is released on cleavage of the scissile bond rema ins anchored in its original position, inhibiting the attack of water on the acyl-enzyme that is formed between the protease and the cleaved inhibitor. More importantly, the NH2 group is optimally placed for re versing the formation of the acyl-enzyme so that the equilibrium betwe en the cleaved and uncleaved inhibitor, bound to the protease, greatly favors the uncleaved complex.