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

H-1 and C-13 NMR spectra of trypsin and ribonuclease, stabilized by ch emical modification with a hydrophilic polymer, have been obtained ove r a wide pH range (1-11). The spectral features, referred to some nucl ei of the catalytic sites (the ''catalytic triad'' for trypsin and the His-12-His-119 pair for ribonuclease), have been identified using dif ferent NMR techniques as well as chemical modification with selective reagents. It is found that monoprotonation of these systems leads to s ymmetrical (or quasi-symmetrical) H-bonds formed between the basic gro ups. This allows us to explain the discrepancies between experimental data obtained by different authors on the protonation sites in these c atalytic systems. The simulation of the catalytic triad by a N-15 labe led low molecular weight model has led us to the conclusion that exter nal agents do not cause any discrete proton transfers but do cause a s mooth shift of the bridging protons from one basic atom to another, wi th the quasi-symmetrical H-bonds being formed in intermediate cases. O n the basis of these experimental data, a new concept has been propose d for the mechanism of acid base catalysis performed by the pairs of w eak basic groups like His-Im and Asp(Glu)-COO- (pK(a) 3-7) which are n ot 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 th e other hand, of facilitating the free movement of a proton in the fie ld of several basic atoms when going along the reaction coordinate. Th e energy of the very strong H-bonds thus formed diminishes the activat ion energy of the reaction.