Variation in aspects of cysteine proteinase catalytic mechanism deduced byspectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations

The possibility of a slow post-acylation conformational change during catal ysis by cysteine proteinases was investigated by using a new chromogenic su bstrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain , caricain, actinidin and ficin), and stopped-flow spectral analysis to mon itor the presteady state formation of the dithioacylenzyme intermediates an d their steady state hydrolysis. The predicted reversibility of acylation w as demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substr ates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S-2-subsites, whereby that of ac tinidin in the free enzyme is shorter blue to the presence of Met(211). Mol ecular dynamics simulation suggests that during substrate binding the sidec hain of Met(211) moves to allow full access of a Phe sidechain to the S-2-s ubsite. The highly anionic surface of actinidin may contribute to the speci ficity difference between papain and actinidin. During subsequent molecular dynamics simulations the P-1 product, methanol, diffuses rapidly (over < 8 ps) out of papain and caricain but 'lingers' around the active centre of a ctinidin. Uniquely in actinidin, an Asp(142) -Lys(145) salt bridge allows f ormation of a cavity which appears to constrain diffusion of the methanol a way from the catalytic site. The cavity then undergoes large scale movement s (over 4.8 Angstrom) in a highly correlated manner, thus controlling the m otions of the methanol molecule. The changes in this cavity that release th e methanol might be those deduced kinetically.