THE ROLE OF SHORT HYDROGEN-BONDS IN MECHANISMS OF ENZYMATIC ACTION

H-1 and C-13 NMR spectra of trypsin and ribonuclease, stabilized by chemical modification with a hydrophilic polymer, have been obtained over a wide pH range (1-11). The spectral features, referred to some nuclei of the catalytic sites (the ''catalytic triad'' for trypsin and the His-12-His-119 pair for ribonuclease), have been identified using different NMR techniques as well as chemical modification with selective reagents. It is found that monoprotonation of these systems leads to symmetrical (or quasi-symmetrical) H-bonds formed between the basic groups. This allows us to explain the discrepancies between experimental data obtained by different authors on the protonation sites in these catalytic systems. The simulation of the catalytic triad by a N-15 labeled low molecular weight model has led us to the conclusion that external agents do not cause any discrete proton transfers but do cause a smooth shift of the bridging protons from one basic atom to another, with the quasi-symmetrical H-bonds being formed in intermediate cases. On the basis of these experimental data, a new concept has been proposed for the mechanism of acid base catalysis performed by the pairs of weak basic groups like His-Im and Asp(Glu)-COO- (pK(a) 3-7) which are not capable of proton abstraction from alcoholic or water OH groups (pK(a) > 13). This catalysis may consist on the one hand of changing the charge densities on reacting groups due to strong H-bonding and, on the other hand, of facilitating the free movement of a proton in the field of several basic atoms when going along the reaction coordinate. The energy of the very strong H-bonds thus formed diminishes the activation energy of the reaction.