No abstract

The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of ␣-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.

Abstract␣-Chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular dynamics and targeted molecular dynamics simulations in aqueous environment on the activation and inactivation processes of three different types of chymotrypsin. These are the wild-type bovine chymotrypsin containing the propeptide and the bovine and rat chymotrypsin lacking the propeptide. From these simulations, the importance of the propeptide and of the sequence differences between the rat and bovine variants from the viewpoint of activation could be evaluated and compared with previous fluorescence stopped flow results. The obtained results show the unambiguous influence of the propeptide on the explored conformational space, whereas the sequence differences between bovine and rat chymotrypsin play a minor role. The main features of activation are present in both the wild type and the variant lacking the propeptide, despite the fact that different parts of the conformational space were explored. The comparison of all trajectories shows that particular amino acid residues, such as 17, 18, 19, 187, 217, 218, and 223, undergo large dihedral transitions during the activation process, suggesting a role as hinge residues during the conformational change.Keywords: conformational change; molecular dynamics simulation; targeted molecular dynamics; pathway calculation; chymotrypsinogen; chymotrypsin; A-chain (propeptide); fluorescence stopped flow Supplemental material: see www.proteinscience.org Conformational changes play an essential role in the modulation of biological activity in proteins. Proteases are synthesized as inactive proenzymes, zymogens, and perform a series of conformational changes toward their active conformation in order to provide a site-and time-specific regulation of their proteolytic activity. The inactive protein precursors often consist of the intact protease with an N-terminal extension or prosegment, stabilizing the inactive enzyme conformation. Activation of the enzyme is then accomplished by limited cleavage of the propeptide in the zymogen. A typical example is the activation of chymotrypsinogen to chymotrypsin occurring after tryptic cleavage of the peptide bond between amino acid residues 15 and 16. This cleavage triggers a series of conformational changes that activates the enzyme (Bode et al. 1978;Bode 1979;Wang et al. 1985;Wroblowski et al. 1997). It allows the formation of a salt bridge between the newly released Ile 16 N terminus and the Asp 194 carboxyl group. This activation of the zymogen can also be mimicked by a pH jump from an inactive chymotrypsinogen-like structure at high pH to an active conformation around pH 7 and so can be followed spectroscopically (Fersht and Renard 1974 Abbreviations: rms, root mean square; MD, molecular dynamics; rmsd, root mean square deviation; TMD, targeted molecular dynamics; ⌬A-chymotrypsi...

No abstract