Comparative Kinetic Properties of α, β and Ψ Forms of Trypsin

Abstract

The kinetics of ester substrate hydrolysis catalysed by the a, j, and $ forms of trypsin have been compared. An affinity chromatography method for +-trypsin purification is described. At optimum pH, with esters as substrates, there is no significant difference between the activity of the a and j forms; j-trypsin, however, is about 40 % more active than a-trypsin on benzoyl-L-arginine amide.At acidic pH, (= 3.6), j3-trypsin is more reactive toward p-nitrophenyl-p'-guanidino benzoate HCl than a-trypsin, and differential active-site titrations of the a and fi forms can be carried out in mixtures of both of them. Hydrolysis of specific esters by $-trypsin is very slow compared to native trypsin but it is not completely aspecific. Carbobenzoxy-L-lysine benzyl ester is a relatively good substrate of this enzyme form; k,,, values for $-trypsin are very sensitive to the substrate leaving group, in contrast to that which is observed with native trypsin. Furthermore, experiments involving nucleophilic competition indicate that the acylation step becomes rate-limiting for the hydrolysis of cationic ester substrates by $-trypsin.It is concluded that in tryptic catalysis enzyme acylation is a more specific step than substrate binding or enzyme deacylation. The acylation of the enzyme is greatly perturbed both by substrate modifications and by structural alterations of the native enzyme molecule. The role of the essential active-site carboxylic group (Asp-177) is discussed. It is proposed that its function in tryptic catalysis is to decrease the activation free energy for enzyme acylation instead of stabilizing the initial enzymesubstrate complex.The kinetic and structural properties of trypsin, a proteolytic enzyme, have been studied extensively [l -31. Tryptic catalysis is specific for peptide, amide and ester bonds formed with the carboxylic function of the basic amino acids lysine and arginine [4,5]. Tryptic specificity is not absolute, however, and some non-basic amino acid ester derivatives are also substrates of this enzyme [6,7].The kinetics of tryptic hydrolysis of synthetic ester substrates can be described by a three-step mechanism involving an acyl-enzyme intermediate (ES') in addition to the classical Michaelis complex (ES), as given in Eqn (1) :+ PI where PI and P, are the reaction products (alcohol and acid, respectively, in the case of an ester substrateAbbreviations. Amino-acid derivatives are abbreviated according to the general rules of IUPAC-IUB, Commission on Biochemical Nomenclature, see Eur. J. Biochern. 27, 9 . 201-207 (1972).Enzyme. Bovine trypsin (EC 3.4.4.4).In a previous paper [8] we analysed and discussed tryptic specificity in terms of the formalism of Eqn (1). The following conclusions were reported.a) The deacylation step (k,) is rate-limiting for specific ester substrates (lysine or arginine derivatives).b) The acylation step (k,) is at least partly ratelimiting for nonspecific ester substrates (tyrosine, phenylalanine and glycine derivatives). c) Structural modifications of a specific substra...

Improved purification process of β- and α-trypsin isoforms by ion-exchange chromatography

“…Determination of specific activities of commercial and purified trypsin isoforms was made using BApNA (Erlanger, et al, 1961), and the results are shown in fig.4. The results are in accordance to Foucault (Foulcault, et al, 1974a andFoulcault, et al, 1974b). The low specific activity found with commercial trypsin is due to factors unstabilizing the sample, as peptides inhibitors in the commercial trypsin preparation, storage time or storage at very low temperatures, possibly leading to cold denaturation.…”

Spectroscopic and thermodynamic properties of Debaryomyces hansenii UFV-1 α-galactosidases

“…Although the D. hansenii UFV-1 extracellular and intracellular ␣-galactosidases had presented similar properties, as molecular mass (by MALDI-TOF and SDS-PAGE), N-terminal amino acid sequences [16], and secondary structures, thermodynamic and kinetic significant differences showed that the ␣-galactosidases (extra-and intracellular) exist as two isoforms. Similar results were found for other well studied proteins, such as the trypsin isoforms [37,38].…”

“…Thus, the calorimetric analysis supply more reliable thermodynamic data, therefore the calorimetric measured is resultant of direct thermal effects of protein denaturation. This data complement the spectroscopic structural studies, supplying a more complete description of the system (protein + carbohydrate + solvent) [20,38].…”

Histidine mapping of serine protease: a synergic study by IMAC and molecular modelling

“…Trypsin activity was assayed using BAEE as described by Schwert and Takenaka (1955). The method of Foucault et al (1974) with BAA and BAEE was used to discriminate the a and b forms of trypsin. Subtilisin activity was followed using TAME as described by Rick (1974).…”