THE TRYPSIN CATALYZED HYDROLYSIS OFp-NITROPHENYL ACETATE

Abstract

The hydrolysis of p-nitrophenyl acetate (NPA) by trypsin has been investigated in the early stage of the reaction using stopped-flow techniques. The influence of pH on the initial rate suggests competitive inhibition of the active site of the enzyme by hydrogen ions. The dissociation constant of the enzyme obtained from the kinetics of this reaction (pK = 6.9) indicates possible catalysis by an amino group or an imidazole group of the enzyme. Lysine methyl ester as a n analogue of the enzyme catalyzes the hydrolysis of NPA under similar experimental conditions. The results are described in terms of an assumed mechanism and the nature of the catalytic site is discussed.Hartley and Kilby (1, 2) first reported t h a t the enzyme chymotrypsin was capable of catalyzing the hydrolysis of p-nitrophenyl acetate (NPA). From their results they concluded that the mechanism consisted of three distinct stages. The first stage is extremely rapid and concerns the combination of enzyme with substrate, the second stage is moderately rapid and involves the liberation of phenol and the acetylation of the enzyme, while the last stage, which is slow, liberates acetate with the consequent regeneration of enzyme.Gutfreund and Sturtevant (3), employing stopped-flow techniques, showed that the rate constant for acetylation (second stage) of chymotrypsin with NPA was p H independent. This is contrary to their results for dinitrophenyl acetate (4) and the results of other workers (5) who report p H dependence.The purpose of our investigation into the mechanism of trypsin catalyzed hydrolysis of NPA is mainly to clarify the problem as to whether or not the rate constant related to acetylation is p H inclependent, and compare the suggested mechanism with a mechanism recently proposed by Cunningham (6). His scheme, which emphasizes the hydroxyl group of a serine residue as primary site rather than the imidazolyl group of histidine, satisfies the available experimental evidence (7). However, when trypsin (8) or chymotrypsin (9) are treated with DFP3"diisopropyl fluorophosphate) no histidine residue occurs in the vicinity of the phosphorylated serine, so t h a t we would like to present some evidence that an amino group other than the imidazolyl might function as part of the catalytic site. E X P E R I M E N T A I LTwice crystallized trypsin (salt-free) was obtained from the Mann Research Laboratories Inc., New York. T h e substrate p-nitrophenyl acetate was synthesized as described by Chattaway (10). All reactions were carried out in 20% (v/v) isopropyl alcohol a t 25" C with 0.025 M phosphate as buffer. T h e p H values quoted refer to the buffer and were measured in the absence of alcohol, but in the presence of enzyme, using a Beckman Model G p H meter.T h e rapid acetylation reaction was studied employing the stopped-flow apparatus 'Manz~script received A z~g z~s t 22, 1968.

The Reactivation of Diethylphosphoryltrypsin*

“…6). This is in accord with the data reported by Stewart and Ouellet (1959 j for the deacetylation of monoacetyltrypsin and also agrees with the findings of Bendei et al (1961) for the deacylation of cinnamoyl-a-chymotrypsin. On the other hand, the rate of spontaneous reactivation of Sarin-inactivated chymotrypsin was reported to occur more rapidly as the pH was lowered (Green and Nicholls, 1959).…”

KINETICS OF THE CATALYZED HYDROLYSIS OF p-NITROPHENYL ACETATE

“…T h e same result was found in t h e present work, but the rate constant was significantly smaller, as shown in Table I. T h i s difference is believed to be due to the presence in the previous work2 of peroxides, as lThis possibiCity is rcferred to i n footnote 16 contaminants in the dioxane solvent; peroxides are known to have a powerful catalytic effect. The previous study in dioxane (9) also showed excessive hydroxide ion catalysis, attributed to the same cause.…”

Catalytic Activity of Trypsin with an Acylated “Active” Serine

“…The rate limiting step is deacylation and it can be slowed down by lowpH [16].It was established that p-nitrophenylacetate specifically acetylates the "active" serine in a-chymotrypsin [17,18]. Taking into account the structural analogy between trypsin and a-chymotrypsin [19], the similarity of kinetic constants for the cleavage of p-nitrophenyl acetate by both enzymes [16] we can admit, that the quasisubstrate will selectively acetylate at low p H practically the "active" serine of trypsin. This conclusion is confirmed by the data on titration of the active centers of native trypsin [20] by p-nitrophenyl acetate or the specific substrate p-nitrophenyl ester of Nu-carbobenzoxy-L-lysine.…”