Backbone Dynamics of Chymotrypsin Inhibitor 2: Effect of Breaking the Active Site Bond and Its Implications for the Mechanism of Inhibition of Serine Proteases

Abstract

The backbone dynamics of uniformly 15N-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 15N longitudinal (T1) and transverse (T1 rho) relaxation times and (1H)-15N NOE enhancements has been measured for all backbone NH groups in both proteins. Information on internal motions has been extracted from these data using the model-free approach to determine order parameters (S2) and effective internal correlation times (tau e). The data indicate that most of the backbone of CI2 is highly constrained (S2 approximately 0.9) with the exception of residues in the binding loop (residues 54-64), which have slightly lower order parameters. Most of the residues in the CI2(20-59).(60-83) complex are also highly constrained (S2 approximately 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 first half of the loop region have significantly lower order parameters than those in the second half of the loop. This observation suggests that the NH2 group that is released on cleavage of the scissile bond remains 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 reversing the formation of the acyl-enzyme so that the equilibrium between the cleaved and uncleaved inhibitor, bound to the protease, greatly favors the uncleaved complex.

Direct Observation of Microscopic Reversibility in Single-molecule Protein Folding

“…Experimentally, the active site loop and the N-terminal tail comprise the most dynamic portions of the native protein. 35,36 As would be expected, the refolding to N' yields large deviations from the 398 K denatured state (Figure 1). …”

A clogged gutter mechanism for protease inhibitors

“…3B). There has been substantial NMR evidence that this network remains intact in the cleaved inhibitor (examined in the absence of enzyme), and stabilizes the newly formed N terminus (19). Similar interactions have been identified in cleaved Cucurbita maxima trypsin inhibitor V, an inhibitor with sequence and structural homology to CI2 (33)(34)(35).…”

“…1-3: Reasons postulated for the inhibitors' surprising lack of reactivity include (i) that the extreme rigidity of the complex prevents productive nucleophilic attack (9)(10)(11)(12)(13)(14)(15), (ii) that poor orientation of the reacting groups results in a nonproductive complex (16)(17)(18), and (iii) that positioning of the leaving group H 2 N-R 2 in the acyl-enzyme complex favors the back reaction toward the Michaelis complex (12,14,19). To clarify this fundamental anomaly of enzyme catalysis, we initiated studies involving incubation of the classical serine protease subtilisin (EC 3.4.21.62) with a classical inhibitor, chymotrypsin inhibitor 2 (CI2).…”

“…One hypothesis suggested by Fersht and coworkers (12,19) is that the leaving group amine is poised for nucleophilic attack on the acyl-enzyme, resulting in the back reaction illustrated by k Ϫ2 in Eq. 2.…”

A New Generic Method for the Production of Protein-Based Inhibitors of Proteins Involved in Cancer Metastasis

“…We also wanted a protein with a straight-forward activity assay so that we could use high-throughput methods to monitor the consequences of mutation. Eglin c is a proteinase inhibitor that acts by binding so tightly to its target in the Michaelis complex that few eglin molecules make it into the transition state (48). The proteinase binding site is contained within a ten amino acid loop that is on the opposite side of eglin from the ahelix that contains the mutated residues in our libraries (Figure 1).…”