Abstract
Various mechanisms for the reversible formation of a covalent tetrahedral complex (TC) between papain and peptidyl aldehyde inhibitors were simulated by DFT calculations, applying the quantum mechanical/self consistent reaction field (virtual solvent) [QM/SCRF(VS)] approach. Only one mechanism correlates with the experimental kinetic data. The HisβCys catalytic diad is in an N/SH protonation state in the noncovalent papainβaldehyde Michaelis complex. His159 functions as a general base catalyst, abstracting a proton from the Cys25, whereas the activated thiolate synchronously attacks the inhibitor's carbonyl group. The final product of papain inhibition is the protonated neutral form of the hemithioacetal TC(OH), in agreement with experimental data. The predicted activation barrier g = 5.2 kcal mol^β1^ is close to the experimental value of 6.9 kcal mol^β1^. An interpretation of the experimentally observed slow binding effect for peptidyl aldehyde inhibitors is presented. The calculated g is much lower than the rate determining activation barrier of hemithioacetal formation in water, g, in agreement with the concept that the preorganized electrostatic environment in the enzyme active site is the driving force of enzyme catalysis. We have rationalized the origin of the acidic and basic p__K__~a~'s on the k~2~/K~S~ versus pH bellβshaped profile of papain inhibition by peptidyl aldehydes. Proteins 2011. Β© 2010 WileyβLiss, Inc.