Trypsin Catalysis Research Articles

Substrate specificity of insect trypsins and the role of their subsites in catalysis

Trypsins have high sequence similarity, although the responses of insect trypsins to chemical and natural inhibitors suggest they differ in specificities. Purified digestive trypsins from insects of four different orders were assayed with internally quenched fluorescent oligopeptides with two different amino acids at P1 (Arg/Lys) and 15 amino acid replacements in positions P1′, P2′, P2, and P3. The binding energy ( Δ G s , calculated from K m values) and the activation energy ( Δ G T ‡ , determined from k cat / K m values) were calculated. Dictyoptera, Coleoptera and Diptera trypsins hydrolyze peptides with Arg at P 1 at least 3 times more efficiently than peptides with Lys at P 1, whereas Lepidoptera trypsins have no preference between Arg and Lys at that position. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results suggested that insect trypsin subsites become progressively more hydrophobic along evolution. Apparently, this is an adaptation to resist plant protein inhibitors, which usually have polar residues at their reactive sites. Results also suggested that, at least in lepidopteran trypsins, S3, S2, S1′, and S2′ significantly bind the substrate ground state, whereas in the transition state only S1′ and S2′ do that, supporting aspects of the presently accepted mechanism of trypsin catalysis. Homology modeling showed differences among those trypsins that may account for the varied kinetic properties

The enhancement of electrostriction caused by lowering the solvent dielectric constant leads to the decrease of activation energy in trypsin catalysis

The contribution of electrostriction of the solvent to the stabilization of the negatively charged tetrahedral transition state of a trypsin-catalyzed reaction was probed by means of kinetic studies involving high-pressure and solvent dielectric constant. A good correlation was observed between the increased catalytic efficiency of trypsin and the decreased solvent dielectric constant. When the dielectric constant of the solvents was lowered by 4.68 units, the loss of activation energy and that of free energy of activation were 2.26 kJ/mol and 3.09 kJ/mol, respectively. The activation volume for k cat decreased significantly as the dielectric constant of the solvent decreased, indicating that the degree of electrostriction of the solvent around the charged tetrahedral transition state has been enhanced. These observations demonstrate that the increase in the catalytic efficiency of the trypsin reaction with decreasing dielectric constant resulted from the stabilization of electrostatic energy for the formation of an oxyanion hole, and this stabilization was caused by the increase of electrostricted water around the charged tetrahedral transition state. Therefore, we conclude that control of the solvent dielectric constant can stabilize the tetrahedral transition state, and this lowers the activation energy.

Functional Word in a Protein. III. Syntax and Energetics.

Trypsin catalysis that was analyzed by the DEV model is discussed from the standpoint of energetics. In trypsin, the nucleophilic activity of 177-S is enhanced by interaction with 40-H and 84-D, and the steric strain that will be caused by many kinds of structural fluctuation is gradually accumulated and stored as the internal energy of trypsin. A small amount of energy exerts the each fluctuation, which arises in a mild physical condition. We call this process a unit process. Such unit process consequently leads to the accumulation of free energy as well as to the activation of enzymatic reaction. The sophisticated nature of bio-catalysis is also discussed.

The ability of time-resolved Laue crystallography to discriminate small changes in structure: appearance of a hydrolytic water molecule in trypsin catalysis

The ability of time-resolved Laue crystallography to discriminate small changes in structure: appearance of a hydrolytic water molecule in trypsin catalysis

Laue diffraction as a tool in dynamic studies: hydrolysis of a transiently stable intermediate in catalysis by trypsin

A transiently stable intermediate in trypsin catalysis, guanidinobenzoyl-Ser-195 trypsin, can be trapped and then released by control of the pH in crystals of the enzyme. This effect has been investigated by static and dynamic white-beam Laue crystallography. Comparison of structures determined before and immediately after a pH jump reveals the nature of concerted changes that accompany activation of the enzyme. Careful analysis of the results of several structure determinations gives information about the reliability of Laue results in general. A study of multiple exposures taken under differing conditions of beam intensity, crystal quality, and temperature revealed information about ways to control damage of specimens by the X-ray beam.

Probing trypsin specificity with basic 3-substituted glutarate diesters and related monoester substrate analogs: Evidence for allosteric activation of trypsin catalysis

A series of dimethyl glutarates bearing basic substituents at C-3, and related monoesters, have been evaluated as substrates of trypsin in order to probe the asymmetric synthetic potential of the enzyme with respect to enantiotopic ester group, and enantiomer, discrimination. While none of the mono- or diesters proved to be a trypsin substrate, several of them accelerated trypsin-catalyzed hydrolysis of the standard reference substrate BAEE, in a manner consistent with an allosteric activation process. The results provide the first examples of allosteric activation of trypsin by modifiers that are sterically precluded from interacting effectively at the acive site.

Ab initio study on the transition state of acylation step of trypsin catalysis.

The transition state of acylation step of trypsin catalysis was determined by molecular orbital calculations. The calculations were carried out at the RHF-LCAO-SCF approximation level with double zeta basis set (plus polarization functions). The role of His57 residue in the acylation step of the catalytic reaction of trypsin was analysed from a quantum mechanical point of view. The influences of surrounding residues, such as oxyanion hole and Asp102-, and the electrostatic effect of the other regions of the enzyme were also studied. His57 was proved to capture the proton from Ser195 side chain terminus with its lone pair and to transfer it to substrate with electrostatic assistance of Asp102- and oxyanion hole.

Action of urokinase and trypsin on angiotensin, ACTH and the oxidized B chain of insulin

N-terminal analysis of the products of hydrolysis of angiotensin, ACTH and the oxidized B chain of insulin after 4 h incubation with trypsin and urokinase reveals a great qualitative similarity in the action of the two enzymes. As expected, the rates of hydrolysis differ significantly and are much higher in the case of trypsin catalysis than in the case of urokinase catalysis. Unexpectedly, however, a decrease in the difference between the catalytic activity of the two enzymes, by increasing the number of Arg and Lys residues present in the substrate, has been observed. Trypsin Urokinase specificity HPLC enzyme kinetics Angiotensin ACTH Oxidized insulin B chain

Kinetics of trypsin catalysis in the industrial conversion of porcine insulin to human insulin.

The kinetics of coupling and transpeptidation reactions catalysed by trypsin were studied in media with low water contents to see whether the usual Michaelis-Menten kinetics applied under the unusual conditions used in peptide bond synthesis, to obtain information about the magnitude of rate constants and activation energies, and to gain insight into the mechanism of catalysis. Porcine insulin, des-(AlaB30)-porcine insulin, human insulin-ThrB30-OMe and human insulin-ThrB30(But)-OBut were used as substrates. Two threonine esters (Thr-OMe and Thr(But)-OBut) were used for transpeptidation and coupling. The reactions progressed according to first-order kinetics until about 35% conversion, and the experimental data were adequately explained by Michaelis-Menten kinetics. The rates of the coupling and transpeptidation reactions in media with low water contents were orders of magnitude below the rates of peptide bond hydrolysis by trypsin in water. It was not possible to approach saturation of the enzyme with substrate so determination of Km was impossible, but for each substrate a value larger than 0.1 M was estimated from the Lineweaver-Burk plot. The rate of release of alanine from porcine insulin depended on the type of threonine ester present; for example, Thr-OMe inhibited the reaction. Coupling occurred faster than transpeptidation. However, in the medium used, the activation energies for the two reactions were similar (about 50 kJ/mol), so the difference in reaction rates is probably due to different transmission coefficients in the activated transition states. Computer simulations enabled us to obtain quantitative descriptions of the reaction progress curves from fitted rate constants.

The mechanism of trypsin catalysis at low pH. Proposal for a structural model.

The mechanism of trypsin catalysis at low pH. Proposal for a structural model.

The detection, accumulation and stabilization of a tetrahedral intermediate in trypsin catalysis

The reaction of trypsin with N α-carbobenzoxy-L-lysine p-nitroanilide has been studied in the 0 to −60°C temperature region, over a wide range of pH ★ values, using aqueous-dimethyl sulfoxide cryosolvent. Spectrophotometric and kinetic evidence for the detection and stabilization of a tetrahedral intermediate is presented. The rate of formation of this intermediate, and the concentration accumulated, were pH ★-dependent. At pH ★ ≤ 6 (< −40°C) essentially no tetrahedral adduct is formed or accumulated. Instead another intermediate, resembling the substrate in its spectral properties, is detected.

Effect of methylamine on trypsin catalysis.

The effect of the basic group of specific substrates of trypsin on the kinetics of the enzyme was studied. The basic group was substituted by methylamine added to a non‐specific substrate. The kinetic constants of tryptic hydrolysis of N‐acetyl‐l‐alanine ethyl ester involving methylamine, at pH 6.3–8.5 were determined. For comparison, the constants of trypsin and α‐chymotrypsin were also determined.It can be concluded that methylamine increases the rate of hydrolysis catalyzed by trypsin. The relative increase of the pH‐independent limiting value of (kc/Km)max is shown to be fivefold and the pKa decreases by 0.4 unit. The increase of kc is nearly fourfold. The rate constants of the hydrolysis steps catalyzed by trypsin and trypsin · methylamine, respectively, were assayed by means of the nucleophile 1,4‐butanediol at pH 6.6. There is a sixfold increase of the original rates caused by methylamine for acylation (k2) and a threefold increase for deacylation (k3) but the substrate dissociation constant (Ks) was almost unchanged.A comparison of the individual rates of the methylamine‐activated tryptic hydrolysis of nonspecific substrates with the appropriate data of the trypsin‐catalyzed hydrolysis of specific substrates indicates that there is an approximately 1000‐fold difference between the rate constants for the acylation step, while in deacylation the difference is insignificant.The methylammonium ion bound to trypsin is supposed to keep the enzyme through the catalysis in a more active conformation than the original one. This indirect effect might bear only a small part of the trypsin‐substrate interaction.

Striking susceptibility of a kinin-yielding pentadeca-peptide to tryptic hydrolysis

Hydrolysis of Lys-Arg-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser by trypsin (EC 3.4.21.4) yields lysyl-bradykinin by rupture of the Arg-Ser bond. The k cat/K m value found for this hydrolysis was 1.4 × 10 10 M −1 × sec −1, which is 10 −5-fold higher than that obtained for the hydrolysis of bradykinyl-Ser-Val-Gln-Val-Ser. This effect was abolished by acetylation of the lysine amino groups of the pentadecapeptide. Contrarywise, the esterolytic activity of trypsin on bradykinin methyl ester was the same as in lysyl-bradykinin methyl ester. The high susceptibility of Lys-bradykinyl-Ser-Val-Gln-Val-Ser to trypsin catalysis is striking because: a) it constitutes the first example that an amino acid residue distant from the bond split may enhance trypsin catalysis; b) this pentadecapeptide is the best synthetic substrate so far described for trypsin and c) the value of k cat/K m for its hydrolysis is unusually high for proteases.

The significance of an unique pH-profile for trypsin catalysis

The pH-profiles for the trypsin-catalyzed hydrolysis of non-N-acylated α-amino substrates were found to be complex. The profile for L-lysine methyl ester gave two rate constants and four acid-base equilibrium constants. The rate constants arise from two different ionic forms (structures) of the site which are reactive. The pKa values calculated from the profile are indicative of an ionizable amino, imidazoyl and carboxyl group at the site, and the free α-amino group in the substrate. A negative heat of ionization was obtained for the group with the smallest pKa, which is kinetic evidence for the existence of a free carboxyl at or near the site.

PH Effects in trypsin catalysis

A kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at pH values ranging from 6 to 10. The substrate concentrations varied from 1.7 × 10−3 to 4.3 × 10−2 M. From the rates were calculated, at each pH, values of [Formula: see text] (corresponding to [Formula: see text]), [Formula: see text] (corresponding to [Formula: see text]) and [Formula: see text] The specific levorotation of trypsin was measured and found to vary with pH in the pH region 5–11, the change in specific rotation following the ionization of a single group with pK(app) of 9.4. At pH 11 the specific rotation of trypsin, its zymogen, and its phosphorylated derivative were approximately the same, suggesting similar conformations for all three forms of the protein.The kinetic results on the acid side were very similar to those obtained by other investigators for chymotrypsin; they imply that there is a group of [Formula: see text] in the free enzyme, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, which can only occur if it is unprotonated. The behavior on the basic side was found to be different from that with chymotrypsin revealing a decrease in [Formula: see text] at high pH corresponding to a value of [Formula: see text] whereas [Formula: see text] showed sigmoid pH-dependence. An interpretation of these results that is consistent with all available information is that a group of [Formula: see text] (presumably the —NH3+ function of the terminal isoleucine) controls the conformation and thereby the activity of the enzyme at different stages of complex formation. In contrast to chymotrypsin, the pK of this ionizing group appears to be generally lowered by covalent complex formation between trypsin and its substrates.

The effect of N-bromosuccinimide upon trypsinogen activation and trypsin catalysis

Modification of trypsinogen with N-bromosuccinimide (NBS) significantly alters the appearance and the time required for the development of the activation difference spectrum. The kinetic properties of the activated modified trypsinogen (ATGM) were compared with similarly modified trypsin (TM). Neither ATGM nor TM showed significant changes in the binding parameters K s,app and K i evaluated for mixture of l-arginine methyl ester (L-AM) and benzamidine. There was a sharp decrease in k app for ATGM with increasing extent of NBS modification. This apparent decrease in the catalytic efficiency can be explained by a loss in active site concentration, based upon inhibition measurements with soybean trypsin inhibitor. These results were in contrast with those for modified trypsin, where there was a decrease in k app with increasing extent of NBS modification, but no loss in active site concentration until the ratio of moles NBS per mole protein had exceeded 2:1. Additional kinetic characterization of the ATGM and TM with tosyl- l-arginine methyl ester ( l-TAM) and activation of chymotrypsinogen gave results which paralleled those for k app with l-AM. Both ATGM and TM exhibited substrate activation with l-TAM.

Effect of alkylguanidines and alkylamines on trypsin catalysis.

Effect of alkylguanidines and alkylamines on trypsin catalysis.

The Mechanism of the Specificity of Trypsin Catalysis: II. COMPARISON OF TRYPSIN AND α-CHYMOTRYPSIN IN THE NONSPECIFIC CATALYSES OF THE HYDROLYSIS OF ACETYLGLYCINE ETHYL ESTER

The Mechanism of the Specificity of Trypsin Catalysis: II. COMPARISON OF TRYPSIN AND α-CHYMOTRYPSIN IN THE NONSPECIFIC CATALYSES OF THE HYDROLYSIS OF ACETYLGLYCINE ETHYL ESTER

The Mechanism of the Specificity of Trypsin Catalysis: III. ACTIVATION OF THE CATALYTIC SITE OF TRYPSIN BY ALKYLAMMONIUM IONS IN THE HYDROLYSIS OF ACETYLGLYCINE ETHYL ESTER

The Mechanism of the Specificity of Trypsin Catalysis: III. ACTIVATION OF THE CATALYTIC SITE OF TRYPSIN BY ALKYLAMMONIUM IONS IN THE HYDROLYSIS OF ACETYLGLYCINE ETHYL ESTER

The Mechanism of the Specificity of Trypsin Catalysis: I. INHIBITION BY ALKYL AMMONIUM IONS

The Mechanism of the Specificity of Trypsin Catalysis: I. INHIBITION BY ALKYL AMMONIUM IONS