Protonic Reorganization in Catalysis by Serine Proteases: Acylation by Small Substrates

Article abstract of DOI:10.1021/ja00536a040

The pH (pD)-rate profiles for acylation of α-lytic protease in protium and deuterium oxides by p-nitrophenyl acetate show pK values of 5.92 and 6.60, well below the enzyme ionization pK values of 6.70 and 7.35.This is attributed to a pH-induced change in the rate-determining step.The data are consistent with an initial acylation of active-site histidine (protolytically assisted, k_H/k_D = 2.4), followed by an intramolecular N -> O acyl shift to active-site serine by parallel specific-acid-catalyzed (k_H/k_D = 0.5) and general-acid-catalyzed (k_H/k_D = 2) routes.The magnitude of pK(D2O) - pK(H2O) and a proton inventory of the general-acid-catalyzed N -> O acyl shift both suggest that deprotonation of α-lytic protease generates an unusual protonic site with a "loosely bound" proton.The β-deuterium isotope effect, k_3H/k_3D = 0.98, for the same step confirms nucleophilic interaction at carbonyl in the transition state.An abbreviated proton inventory for acylation of α-chymotrypsin by p-nitrophenyl acetate is consistent with a "loosely bound" proton there also.A proton inventory for acylation of elastase by N-(carbobenzyloxy)-L-alanine p-nitrophenyl ester is linear, suggesting one-proton catalysis and indicating that if "loosely bound" reactant-state protons are present, they are catalytically silent.The general picture, from this work and that of others, is that the catalytic response of serine proteases to small, "unnatural" substrates is highly variable, both in site of nucleophilic attack and involvement of protolytic catalysis.Probably mutual transition-state interactions over an extended region of both enzyme and natural-substrate structure are required to bring into active function the full catalytic capability with which the serine proteases have been endowed by biological evolution.