The applicability of the acylenzyme hypothesis to the mechanism of tryptic hydrolysis of amide and peptide bonds was studied. The "active" serine of trypsin was acylated with radicals of different size. If the acylenzyme mechanism is valid the kcat of esterase, amidase and peptidase activities of acyltrypsin must always decrease to the same extent. To decrease the degree of deacylation of carboxylic derivatives of trypsin, the enzymatic activity was measured a t 25", pH 6.0 and average incubation time 1 min. As compared with kcat of the native trypsin the values of kcat of esterase and amidase residual activities were found, respectively: for tetraformyl-trypsin 43 and 75O/,, for heptaformyl-trypsin 22 and 55OlO, for monoacetyl-trypsin 6 and 37O/,, for triacetyl-trypsin 1.4 and 22O/,, for monopropionyl-trypsin 9 and 23O/,, for mono-(dimethylpropionyl) -trypsin 12 and 12O/,, for mono-(diethylphosphory1)-or mono-( dimethylphosphory1)-trypsin 1 and lolo. Monoacetyl-trypsin demonstrated 6.401, of esterase and 5S0/, of peptidase activity. On the extrapolation to complete acylation (respectively to the zero esterase activity) the residual amidase activity decreased in the series formyl-, acetyl-, propionyl-trypsin and was equal to zero for dimethylpropionyl-trypsin. The latter acylating radical was similar in its size and hydrophobic nature to the smallest dialkylphosphoryl radicals. It is suggested that the acylenzyme mechanism is at least not a single pathway of the tryptic hydrolysis of amide and peptide bonds.Hydrolases compose a most extensively studied group of comparatively simple enzymes. The aim of this work was to elucidate the intermediate stages of amide hydrolysis catalyzed by trypsin.In the case of hydrolysis of esters, the acylenzyme mechanism is well established [ 1,2] :where E is the enzyme, S-substrate, ES-Michaelis complex, ES'-the acylated enzyme (the ester of the "active" serine in the protein with the acyl moiety of the substrate), PI is the alcohol, P2-the acid derived from the substrate.The mechanism (1) was generalized by many investigators to explain the tryptic hydrolysis of an amide bond and the related peptide bond. The strongest argument in favor of this generalization is the similar behaviour of both hydrolytic reactions of ester and amide cleavage when the "active" serine is phosphorylated by the action of diethyl-p-nitrophenylphosphate or a similar compound. The inhibition constants of both reactions are equal to the rate constant of phosphorylation of the "active" serine in the enzyme [3].This conclusion raises many objections. First of all there are no direct proofs for the applicability of .mechanism (1) for the amide and peptide hydrolysis. Then the acylating power of amides is very low. The blocking of the "active" serine in the enzyme by a bulky hydrophobic diakylphosphoryl group may interact with the hydrolysis of an amide bond by a drastic change of the tertiary structure in the vicinity of the enzyme active center (it is well known, that the conformation of diisopropylphospho...