Computational Study of the Michaelis Complex Formation and the Effect on the Reaction Mechanism of Cruzain Cysteine Protease

Kemel Arafet, Katarzyna Świderek, Vicent Moliner
2018 ACS Omega  
Cruzain, a cysteine protease of the papain family, is essential in the development of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease, making it an attractive target for developing new drugs. The present paper is aimed at the study of the catalytic mechanism of the cruzain by first exploring the different protonation states of the active site Cys25 and His159 in the Michaelis complex and the effect on the full catalytic mechanism of this enzyme. The exploration of the
more » ... librium between these two states has been performed with alchemical free energy perturbation methods with molecular mechanics (MM) force fields and by generating the free energy surfaces in terms of the potential of mean force computed at two levels of theory: AM1d/MM and M06-2X/6-31+G(d,p):AM1d/MM. Alternative mechanisms for the acylation step have been identified on the free energy surfaces and the results suggest the existence of three new reaction mechanisms starting from the peptide binding to the apoenzyme in its neutral Cys25S/His159 dyad state. The mechanism starting with the protonation of the nitrogen atom of the peptide followed by the attack of Cys25S − was revealed as the most favorable one, but it can be competitive with its counterpart mechanism initiated in the Cys25S − /His159H + ion pair Michaelis complex. Analysis of energetic and average geometries will allow continuing improvement of our knowledge on this enzyme at the molecular level, which can be crucial to the design of new inhibitors based on the structures of the transition states (transition states analogues) or stable intermediates.
doi:10.1021/acsomega.8b03010 fatcat:imptnxagrrdkrapghscvbmk4pq