Substrate activation of rat trypsins 1 and 2

Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of K_cat for N¹-tosyl-l-arginine methyl ester vs K_cat for N¹-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N¹-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N¹-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N¹-tosyl-l-arginine methyl ester 4.5 times faster than that of N¹-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.     

The palladium(II) promoted hydrolysis of methyl,ethyl and isopropyl glycylglycylglycinate

The palladium(II) promoted hydrolysis of the ester function of methyl, ethyl and isopropyl glycylglycylglycinate has been studied in detail in the pH range 4–5. The tripeptide esters interact with [PdCl₄]²⁻ at a 1:1 metal-to-ligand ratio to give the complex I in which the α-amino group, two deprotonated amide nitrogen atoms and the alkoxy carbonyl group of the ester act as donors. Rate constants have been determined for hydrolysis of the ester function by water and hydroxide ion and activation parameters obtained for the hydrolysis of the methyl ester. At 25 °C base hydrolysis of the coordinated ester is 10⁶ fold that of the free ester ligand. Mechanisms for the reactions are considered.     

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 × 10¹⁰ 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.     

CRYSTALLINE CHYMO-TRYPSIN AND CHYMO-TRYPSINOGEN : I. ISOLATION,CRYSTALLIZATION, AND GENERAL PROPERTIES OF A NEW PROTEOLYTIC ENZYME AND ITS PRECURSOR

A new crystalline protein, chymo-trypsinogen, has been isolated from acid extracts of fresh cattle pancreas. This protein is not an enzyme but is transformed by minute amounts of trypsin into an active proteolytic enzyme called chymo-trypsin. The chymo-trypsin has also been obtained in crystalline form. The chymo-trypsinogen cannot be activated by enterokinase, pepsin, inactive trypsin, or calcium chloride. There is an extremely slow spontaneous activation upon standing in solution. The activation of chymo-trypsinogen by trypsin follows the course of a monomolecular reaction the velocity constant of which is proportional to the trypsin concentration and independent of the chymotrypsinogen concentration. The rate of activation is a maximum at pH 7.0–8.0. Activation is accompanied by an increase of six primary amino groups per mole but no split products could be found, indicating that the activation consists in an intramolecular rearrangement. There is a slight change in optical activity but no change in molecular weight. The physical and chemical properties of both proteins are constant through a series of fractional crystallizations. The activity of chymo-trypsin decreases in proportion to the destruction of the native protein by pepsin digestion or denaturation by heat or acid. Chymo-trypsin has powerful milk-clotting power but does not clot blood plasma and differs qualitatively in this respect from the crystalline trypsin previously reported. It hydrolyzes sturin, casein, gelatin, and hemoglobin more slowly than does crystalline trypsin but the hydrolysis of casein is carried much further. The hydrolysis takes place at different linkages from those attacked by trypsin. The optimum pH for the digestion of casein is about 8.0–9.0. It does not hydrolyze any of a series of dipeptides or polypeptides tested. Several chemical and physical properties of both proteins have been determined.     

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.     

Rate enhancement by catalytic groups in enzymes. Imidazole catalysis of the hydrolysis of N,O-diacetylserinamide as a model for general base catalysis in chymotrypsin

An attempt to estimate the importance of general acid-base catalysis in enzymic catalysis has been made, using the hydrolysis of the ester group of N,O-diacetylserinamide as a model for the deacylation of acyl-chymotrypsins. General base catalysis of this reaction by imidazole is estimated to reduce the activation energy by at least 31 kJ mol^?1. The rate of reaction, however, is not greatly enhanced because of an unfavourable change in the entropy of activation from ?132 to ?197 JK^?1 mol^?1. At about 300 K, a typical temperature for enzyme-catalysed reactions, the reduction in activation energy would cause a rate enhancement of about 3 × 10⁵-fold if the unfavourable entropy change did not occur. For specific acyl-chymotrypsins the entropy of activation for deacylation is about ?89 J K^?1 mol^?1, allowing the full effect of general base catalysis by imidazole to be realized. It is, therefore, postulated that in the active site of an enzyme, a properly oriented imidazole side chain may catalyse the rate of a reaction 10⁵-fold by general base catalysis.     

N-acetyl-(L-Ala) 3 -p-nitroanilide as a new chromogenic substrate for elastase

The introduction of a useful new chromogenic substrate for the determination of elastase (EC 3.4.4.7) activity is described. N-acetyl-L-Ala-L-Ala-L-Ala-$\text{p}$-nitroanilide (AcAla₃NA) is a new specific elastase substrate whose hydrolysis can be followed spectrophotometrically at 410 nm in a wide pH range. Its rate of hydrolysis by α-chymotrypsin (EC 3.4.4.5) and trypsin (EC 3.4.4.4.) is 0.02% and 0.001% respectively compared to its rate of hydrolysis by elastase. As little as 0.1 μg elastase/ml can be satisfactorily determined. At pH 8, K_m = 0.88 mM and k_cat = 11.9 sec^?1.     

Pancreas acinar cell differentiation. IV. Assay of nanogram levels of chymotrypsin by radioactively labeled synthetic substrate

A procedure for synthesizing ¹⁴C-labeled N-benzoyl-l-tyrosine ethyl ester with the label in the benzoyl group has been described. Using this substrate, which is specific for chymotrypsin, and employing trypsin activation of chymotrypsinogen in an incubation medium containing radiolabeled BTEE, we have presented a method which permits assay of nanogram quantities of chymotrypsin activity in organ-cultured tissue. The radiolabeled product of enzyme hydrolysis, N-benzoyltyrosine, is readily separated by paper chromatography from the unreacted labeled substrate and measured by radioactivity counting.     

The effects of various peptides on the thermotropic properties of phosphatidylcholine bilayers

The effects of an amino acid derivative (N-benzoyl-l-argininamide), four small peptides (Phe-Gly-Phe-Gly, gastrin-related peptide (Trp-Met-Arg-Phe-NH₂), tetragastrin (Trp-Met-Asp-Phe-NH₂), pentagastrin (Boc-βAla-Trp-Met-Asp-Phe-NH₂)) and one medium-sized peptide. glucagon (29 residues), on the gel-to-liquid crystalline transition of a multilamellar suspension of dimyristoylphosphatidylcholine have been studied by means of high-sensitivity differential scanning calorimetry. At low concentrations of added solutes, the temperature at which the excess apparent specific heat in the gel-to-liquid crystalline phase transition of the lipid is maximal is lowered by an amount proportional to the total concentration of the peptide, with proportionality constants ranging from ?0.018 K mM^?1 for Phe-Gly-Phe-Gly to ?3.1 K mM^?1 for the gastrin-related peptide. The lipid mixtures involving the first two solutes listed above exhibited approximately symmetrical curves of excess apparent specific heat vs. temperature. The curves for the other solutes were asymmetric, and could be well represented as the sum of either two or three two-state curves. The asymmetry, which was especially pronounced in the cases of pentagastrin and glucagon, thus appeared to be due to the presence of components having lower and/or higher transition temperatures than that of the lipid. Pentagastrin and glucagon (R.M. Epand and J.M. Sturtevant, Biochemistry 20 (1981) 4603) have much smaller effects on the gel-to-liquid crystalline phase transition of dipalmitoylphosphatidylcholine than on that of the dimyristoyl analog.     

Activation of rat brain adenylate cyclase by proteases: involvement of distinct protein components in the activation by α-chymotrypsin and trypsin

Adenylate cyclase in synaptic plasma membranes from rat brain is activated by α-chymotrypsin or trypsin. These proteases also activate adenylate cyclase reconstituted from the catalytic subunit of adenylate cyclase and the partially purified fraction of the GTP-binding proteins containing both the stimulatory and inhibitory GTP-binding proteins. Properties of the activation of reconstituted adenylate cyclase by the proteases are as follows. (1) The proteases do not directly activate the catalytic subunit. However, the pre-treatment of the partially purified GTP-binding proteins with α-chymotrypsin (100 μg/ml) increases the subsequently reconstituted cyclase activity at least 3-fold. Trypsin (10–30 μg/ml) much more weakly enhances the cyclase activity. (2) α-Chymotrypsin and trypsin synergistically activate the cyclase. (3) Trypsin but not α-chymotrypsin no longer activates the cyclase when the purified stimulatory GTP-binding protein (Gs) replaces the partially purified GTP-binding proteins. (4) The stimulatory effects of α-chymotrypsin and trypsin on the cyclase activity are little or slight unless 5′-guanylylimidodiphosphate (Gpp(NH)p) is present in the reconstitution. (5) The purified βγ-subunits of the GTP-binding proteins markedly inhibit adenylate cyclase. This inhibition is nearly completely attenuated by treating the βα-subunits with α-chymotrypsin (> 10 μg/ml). (6) Trypsin (1–10 μg/ml) inactivates the GTPase of the α-subunit of the inhibitory GTP-binding protein (Gi). This inactivation of the GTPase seems to correlate with the activation of the reconstituted adenylate cyclase by trypsin.We conclude that two distinct protein components are involved in the activation of adenylate cyclase by α-chymotrypsin and trypsin. One component sensitive to α-chymotrypsin is probably the βγ-subunits of the GTP-binding proteins. The other component sensitive to trypsin may be the α-subunit of Gi.     

Effect of external mass transfer of activation energy of hydrolysis of BAEE and casein by trypsin immobilized on molecular sieve type 4A

In this investigation, the activation energies of the hydrolysis of N-(α)-benzol-L -arginine ethyl ester (BAEE) and casein have been determined using trypsin immobilized on molecular sieve type 4A. There is a complete absence of intraparticle diffusion in the system, and the temperature dependence of the reaction has been studied only under external diffusional limitation. While the hydrolysis of BAEE by bound trypsin in found to be controlled by external diffusion, that of casein is kinetically controlled.     

A simple preparation of beta-trypsin based on a calorimetric study of the thermal stabilities of alpha- and beta-trypsin

Bovine trypsin preparations contain, in addition to the single chain form of the enzyme, an active two-chain autolysis product (Schroeder, D. D., and Shaw, E., J. Biol. Chem. (1968), 243, 2943–2949). Differential scanning calorimetric (DSC) studies showed that the single chain form, β-trypsin, is more stable to thermal denaturation than the two-chain form, α-trypsin. Rate constants and activation energies for the thermal denaturation of β-trypsin are 5 × 10^?5 sec^?1 and 69 kcal/mole and of α-trypsin are 5 × 10^?3 sec^?1 and 38 kcal/mole at pH 4.4 and 48 °C. Preparation of pure β-trypsin can be greatly simplified by prior thermal denaturation of the α form. At least 75% of the α form is denatured by heating a 10–15% solution of commercial crystalline trypsin for 30–45 min at 48 °C, pH 4.4, 0.02 m Ca²⁺. The native β-trypsin is then easily isolated from the denatured α-trypsin by batchwise adsorption onto ovoinhibitor-agarose at pH 8. After elution at pH 2, dialysis, and lyophilization an average preparation contained approximately 85% β-trypsin, 10% α-trypsin, and 5% inactive material. Benzamidine was used during the isolation to decrease the rate of conversion of β- to α-trypsin. Because the separation of active β-trypsin from heat-denatured α-trypsin is relatively easy, the total preparation time has been reduced to 1 day.     

Production of L-lysine by immobilized trypsin. Study of DL-lysine methyl ester resolution

The possibility of producing L-lysine from chemically synthesized DL-lysine has been investigated. Optical resolution of racemic DK-lysine may be achieved by using the stereospecific esterasic activity of trypsin on DL-lysine methyl ester, which gives L-lysine and unchanged D-lysine methyl ester. SL-lysine methyl ester spontaneous hydrolysis may be neglected when operating at pH 5.5 and 30 degrees C. Effect of pH and substrate concentration on hydrolysis rate has been investigated when using as a catalyst either soluble or immobilized trypsin. For this purpose, trypsin was coupled onto an amine porous silica, Spherosil, activated with glutaraldehyde. The optimal pH is 5.8 for soluble trypsin and 6.0 for immobilized trypsin. It was yet possible to lower the parent optimal pH of immobilized trypsin, and thus increase its activity at 5.5, by co-grafting onto Spherosil an aminosilane, for enzyme coupling via glutaraldehyde activation and a positively charged diethyl amino ethyl (DEAE) silane, for decreasing the pH of trypsin microenvironment.     

Preparation and use of α-maltosyl fluoride as a substrate by beta amylase

The preparation of pure (amorphous) α-maltosyl fluoride is described. A modification of the procedure of Brauns was used to obtain analytically pure, crystalline hepta-O-acetyl-α-maltosyl fluoride, the structure of which was assigned by¹⁹F-and¹H-n.m.r. spectroscopy. α-Maltosyl fluoride was obtained by deacetylating the heptaacetate. It behaved as a single compound on thin-layer and paper chromatography, and was essentially completely hydrolyzed to maltose and hydrogen fluoride by 0.01M sulfuric acid in 10 min at 100°. Crystalline beta amylase, likewise, catalyzed essentially complete hydrolysis of α-maltosyl fluoride to give maltose and hydrogen fluoride. The rates of hydrolysis catalyzed by beta amylase preparations from sweet potatoes and soybeans acting on a range of concentrations of the substrate produced linear curves for the relationship, 1/v vs 1/S; reaction constants for crystalline, sweet-potato enzyme were K_m 3.6 mM and V_max ~ 2 μ mol/min/mg. The finding that α-maltosyl fluoride is hydrolyzed 30–60 times faster than maltotriose demonstrates for the first time that beta amylase is capable of effecting hydrolysis at an appreciable rate of a substrate having only two d-glucose residues.     

Resistance of α-globulin fromSesamum indicum L. to proteases in relationship to its structure

α-Globulin, the high-molecular-weight protein fraction fromSesamum indicum L., was hydrolyzed to low-molecular-weight protein and peptides by pepsin, while its resistance to hydrolysis by group-specific enzymes, trypsin or α-chymotrypsin, was very high. The protein showed definite structural changes after proteolysis, especially after peptic hydrolysis, as evidenced from various biophysical data. The sedimentation velocity pattern of α-globulin hydrolyzed by trypsin or α-chymotrypsin indicated reduction in the percentage of 11S component, while the pepsinhydrolyzed sample was devoid of any 11S component, indicating the absence of a native protein molecule. The fluorescence emission spectra of the various hydrolyzed α-globulin showed a red shift in the fluorescence emission maximum. The red shift was maximum with α-globulin hydrolyzed by pepsin and minimum with the trypsin-hydrolyzed sample. The far-ultraviolet-circular dichroic measurements indicated that most of the ordered structure of α-globulin was absent after pepsin hydrolysis, while after trypsin and chymotrypsin hydrolysis conformational changes were less.     

Effect of detergents,trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D

The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca²⁺ > Sr²⁺ > Ba²⁺ > Mg²⁺. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me²⁺-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.     

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6?U?ml^?1 (batch cultivation with flask cultivation) or 14.4?U?ml^?1 (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.     

Trypsin cleavage of the α-subunit of beef heart F1-ATPase abolishes ATP synthesis and ATP-driven energy-transduction capabilities

Previous work has shown that mild trypsin treatment eliminates energy-transduction capability and tight (non-exchangeable) nucleotide binding in beef heart mitochondrial F₁-ATPase (Leimgruber, R.M. and Senior, A.E. (1976) J. Biol. Chem. 251, 7103–7109). The structural change brought about by trypsin was, however, too subtle to be identified by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was not defined. In this work we have applied two-dimensional electrophoresis (isoelectric focussing then sodium dodecyl sulfate polyacrylamide gradient electrophoresis) to the problem, and have determined that the α-subunit of F₁ is altered by the mild trypsin treatment, whereas no change was detected in β-, γ-, δ- or ?-subunits. Binding of ADP to the trypsin-treated F₁ was compared to binding to control enzyme over a range of 0–40 μM ADP in a 30 min incubation period. There was no difference between the two enzymes, K^ADP_d in Mg²⁺-containing buffer was about 2 μM in each. Since the tight (nonexchangeable) sites are abolished in trypsin-treated F₁, this shows that tight exchangeable ADP-binding sites are different from the tight nonexchangeable ADP-binding sites. There was no effect of trypsin cleavage of the α-subunit on β-subunit conformation as judged by aurovertin fluorescence studies. The cleavage of the α-subunit which occurred was judged to occur very close to the C- or N-terminus of the subunit and constitutes therefore a small and specific chemical modification which abolishes overall function in F₁ but leaves partial functions intact.     

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate

A method for the determination of elastase activity is described which uses soluble α-[³H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10^?4mN-α-tosyl-l-lysyl chloromethane and 10^?4m tosyl-l-phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.