The stereospecificity of α-chymotrypsin

1. The rates of deacylation of acyl-alpha-chymotrypsins in which the hydrogen-bonding capacity of the acylamino group of the substrate has been systematically removed were measured. 2. The ratio of deacylation rates of l- and d-acyl-enzymes is found to depend largely on the existence in the substrate of an amido -NH- group. 3. The data presented agree with the postulate that the stereospecificity of alpha-chymotrypsin is exercised in catalytic rather than binding steps, and that the active site of the enzyme presents three loci to the substrate: the site containing the catalytic functionalities (including serine-195), the hydrophobic area for amino acid side-chain binding, and a hydrogen-bond acceptor site for acylamino group binding. 4. It is noted that, though the hydrogen-bonding site is crucial for the stereospecificity, the free energy of binding of substrates and inhibitors is dominated by the hydrophobic interaction. 5. It is tentatively proposed that alpha-chymotrypsin selects a high-energy conformation of the substrate when the latter binds at the enzyme's active site.     

S′-subsite mapping of polyethyelene glycol-modified α-chymotrypsin and α-chymotrypsin: a comparative study

Nucleophile specificities of polyethylene glycol-modified α-chymotrypsin and the native enzyme were investigated via acyl transfer reactions using Ac-Tyr-OEt as acyl donor and a large series of peptides and amino-acid amides as nucleophiles. In acyl transfer reactions with amino-acid amines both enzymes prefer basic and bulky amino-acid residues. However, peptides with bulky aliphatic or aromatic residues in P′₁ position were very poor nucleophiles for both enzymes. Surprisingly, peptides having bulky aliphatic or aromatic residues in P′₂ were preferred by the modified enzyme and were apparently more efficient nucleophiles for both enzymes than those with such residues in P′₁. Generally, peptides with a longer chain were weaker nucleophiles in the reactions catalyzed by polyethylene glycol-modified enzyme. In the series of peptides containing a positively charged amino-acid residue in various locations, the order of nucleophilic efficiency is with this location being: P′₁ > P′₃ >P′₂; this is valid for both enzymes.     

Specificity of sweet-almond α-galactosidase

1. The specificity of purified sweet-almond alpha-galactosidase has been investigated with 17 substrates. 2. Some of them exhibited inhibition at high substrate concentrations but others did not. Both substrate types were bound and hydrolysed at the same site on the enzyme. 3. The enzyme is specific for alpha-d-galactosides and beta-l-arabinosides. It did not hydrolyse beta-d-galactosides or alpha-d-glucosides. 4. Among galactosides the order of decreasing rates of enzymic hydrolysis was: aryl alpha-galactosides; sugars; alkyl alpha-galactosides. 5. All substituents in the aryl moiety of aryl alpha-galactosides enhanced V(max.), the electron-releasing (-sigma) groups being more effective than the electron-withdrawing (+sigma) groups. The substituent groups did not alter K(m) appreciably. 6. Implications of these results are discussed from a mechanistic viewpoint.     

The binding of inhibitors to α-chymotrypsin

1. The binding of three competitive inhibitors, N-acetyl-d-tryptophan, N-acetyl-l-tryptophan and N-acetyl-d-tryptophan amide, to alpha-chymotrypsin was studied over the pH range 2.20-9.65 by the technique of equilibrium dialysis. 2. Within the limits of the experimental method, the binding of the uncharged amide inhibitor is independent of pH over the range investigated. 3. The binding of each of the enantiomeric acids is dependent on the ionization of a group on the free enzyme, of apparent pK(a)7.3. 4. It is shown that the ionizing group results in the active site of the enzyme developing a net negative charge above pH7.3. 5. The enzyme groups responsible are tentatively identified, and the significance of the binding constants with respect to the enzymic catalysis is discussed.     

Substrate Specificity of Saccharomyces logos α-Glucosidase

The substrate specificity of Saccharomyces logos α-glucosidase has been investigated.

The enzyme was active especially on maltose and phenyl-α-maltoside. The ratio of hydrolysis for maltose : phenyl-α-maltoside : phenyl-α-glucoside was estimated to be 100:110: 5.5. Therefore, the substrate specificity of the enzyme was quite different from those of other Saccharomyces species, though similar to those of mold α-glucosidases.

Km values for maltose, phenyl-α-maltoside and phenyl-α-glucoside were calculated to be 7.7 mм, 3.6 mм and 8.7 mм, respectively. Of the substrates tested, the enzyme showed a preference for phenyl-α-maltoside.     

Thermostability of α-chymotrypsin encapsulated in reversed micelles

Summary The influence of water content, additives, pH and substrate concentration on the thermostability of -chymotrypsin entrapped in a reversed micellar system of the cationic surfactant TTAB/heptane/ chloroform, was studied. Increasing the water level inside the reversed micelles diminishes the enzyme stability. Enzyme stability enhancement was achieved with the addition of glycerol, by increasing the nucleophile concentration or by decreasing the buffer pH.     

Facile synthesis of L-Dopa esters by the combined use of tyrosinase and α-chymotrypsin

Summary A novel method for the preparative scale synthesis of L-Dopa esters using tyrosinase-catalysed ortho-hydroxylation and proteinase-catalysed transesterification is described. Several L-Dopa esters have been prepared by the combined use of these two enzymes and fully characterised.     

Synthesis and in vitro α-chymotrypsin inhibitory activity of 6-chlorobenzimidazole derivatives

A library of benzimidazole derivatives 1–20 were synthesized, and studied for their α-chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics and molecular docking studies were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor of α-chymotrypsin enzyme (IC₅₀ = 14.8 ± 0.1 μM, K_i = 16.4 μM), when compared with standard chymostatin (IC₅₀ = 5.7 ± 0.13 μM). Compounds 2–8, 15, 17, and 18 showed significant inhibitory activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B, apparently played a key role in the inhibitory potential of the series. Compounds 1–20 were also studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6, 8, 12–14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on the most active members of the series in comparison to the standard compound, chymostatin, to identify the most likely binding modes. The compounds reported here can serve as templates for further studies for new inhibitors of α-chymotrypsin and other chymotrypsin-like serine proteases enzymes.

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The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of α-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH–activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the `microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.     

Effect of aluminum on the binding properties of α-chymotrypsin

 The effect of aluminum ions on the binding properties of α-chymotrypsin has been studied. The results show that aluminum does not affect the catalytic rate constant k _cat, but it acts as an enzyme activator favoring the binding of the substrate to the catalytic site (i.e. decreasing K _m). Furthermore, aluminum binding to α-chymotrypsin displays about a threefold decrease in its affinity for the macromolecular inhibitor bovine pancreatic trypsin inhibitor (BPTI). Altogether, the different effect of aluminum on the binding of synthetic substrates (e.g. N-α-benzoyl-l-tyrosine ethyl ester, BTEE) and macromolecular inhibitors (e.g. BPTI) to α-chymotrypsin suggests the occurrence of an aluminum-linked conformational change in the enzyme molecule which brings about a marked structural change at the primary and secondary recognition sites for substrates and inhibitors. The modulative effect exerted by aluminum on the enzyme hydrolytic activity has been investigated also as a function of pH. The ion-linked effect appears to be dependent on the pH in a complex fashion, which suggests that aluminum binding is controlled by the protonation of at least two classes of residues on the enzyme molecule. Received: 5 December 1996 / Accepted: 11 March 1997     

Exploring the thermal stability and activity of α-chymotrypsin in the presence of spermine

Polyamines such as spermine can have interaction with protein. The aim of the present study was to investigate how spermine could influence the structure, thermal stability, and the activity of α-chymotrypsin. Kinetics, thermodynamics, molecular dynamics (MD), and docking simulations studies were conducted to investigate the effect of spermine on the activity and structure of α-Chymotrypsin (α-Chy) in 50 mM Tris–HCl buffer, with the pH 8, using different spectroscopic techniques as well as molecular docking and MD simulations. The stability and activity of α-Chy were increased in the presence of spermine. The results of the kinetic study showed that the activity of spermine was increased. Enzyme activation was accompanied by changes on the α-Chy conformation. Fluorescence intensity changes showed dynamic quenching during spermine binding. The fluorescence quenching of the α-Chy suggested the more polar location of Trp residues. Near-UV and Far-UV circular dichroism studies also demonstrated the transfer of Trp, Phe, and Tyr residues to a more flexible environment. The increase in the absorption of α-Chy in the presence of spermine was as a result of the formation of spermine–α-Chy complex. Molecular docking results revealed the presence of one binding site with a negative value for the Gibbs free energy of the binding of spermine to α-Chy. Docking study also revealed that van der Waals interactions and hydrogen bonds played a major role in stabilizing the complex.     

Separation,Characterization, and Specificity of α-Mannosidases from Vigna umbellata

Information about the specificity of glycosidase enzymes is important since it affects their use for characterization and synthesis of oligosaccharides. Two α-mannosidases (EC 3.2.1.24), I and II, were isolated from rice beans (Vigna umbellata). The native molecular weight of both isozymes was estimated to be 329,000, but pIs of form I were 5.03-5.34 and pIs of form II were 5.46-6.20. The two isozymes were characterized in terms of optimal pH and temperature, effects of metal ions, inhibition by swainsonine and 1-deoxymannojirimycin, and kinetic parameters for p-nitrophenyl-α-D-mannopyranoside and Manα(1-2)Man. Both enzymes were more specific towards Manα(1-2)Man in both hydrolysis and synthesis, but their hydrolytic specificities towards Manα(1-3)[Manα(1-6)]Man were different.     

Refolding of a denatured α-chymotrypsin and its smart bioconjugate by three-phase partitioning

α-Chymotrypsin inactivated with 8 M urea and 100 mM dithiothreitol could be completely reactivated by subjecting it to three-phase partitioning (TPP). TPP consisted of adding 30% w/v ammonium sulfate and t-butanol (volume equivalent to aqueous solution of denatured α-chymotrypsin) at 25°C. The activated α-chymotrypsin was recovered as an interfacial precipitate between the upper organic and lower aqueous phase. It was found that this could be extended to a thermally inactivated smart bioconjugate of α-chymotrypsin with Eudragit S-100 (a reversibly soluble–insoluble methmethacrylate). The thermally inactivated bioconjugate had to be further subjected to urea and dithiothreitol before refolding by three-phase partitioning. Ninety per cent of the activity of the bioconjugate could be recovered. The free enzyme and its bioconjugate which lost activity in the presence of 90% dioxane recovered 94 and 90% of their activities, respectively, by employing TPP. The refolded free enzyme and its bioconjugate were evaluated in terms of V_max/K_m and their fluorescence emission spectra.     

Anticancer and α-chymotrypsin inhibiting diterpenes and triterpenes from Salvia leriifolia

Salvialeriol (1), a new abietane-type diterpene, was isolated from Salvia leriifolia Benth. (Salvia leriaefolia), along with two known abietane-type diterpenoids, 6-hydroxysalvinolone (2) and deacetylnemorone (3), and two known triterpenes, 2-acetoxylupeol (4), and lupine-2,3-diol (5). Compounds 2–5 are reported here for the first time from this species. Compound 4 was previously reported as a synthetic derivative of 5 and this is the first report of its isolation from a natural source. Compounds 2, 3 and 5 exhibited a potent antiproliferative activity against the prostate cancer cell lines (PC3) with IC₅₀ of 3.9 ± 0.1, 6.2 ± 0.1 and 2.8 ± 0.1 μM, respectively, and cervical cancer cell lines (HeLa) with IC₅₀ of 8.0 ± 0.3, 2.6 ± 0.1 and 2.7 ± 0.1 μM, respectively. Whereas compounds 1 and 4 showed moderate antiproliferative activities against the cell lines. Compounds 1–5 were also evaluated for the inhibition of α-chymotrypsin, a protease enzyme, and 2 exhibited a competitive inhibition of the enzyme (IC₅₀ = 188.8 μM).     

Stability of immobilized α-chymotrypsin in supercritical carbon dioxide

Summary Inactivation of immobilized -chymotrypsin in supercritical carbon dioxide was with a first-order kinetic behaviour. The increase in either the pressure or the temperature of the fluid enhanced the inactivation process of the enzyme. The fluid density was shown as a key parameter on the enzyme stability, enhancing the half-life time proportionally to the physical phase of CO₂, as follows: liquid > supercritical > gas. However, the number of pressurization/depressurization cycles, and the water content of the derivative increased greatly the loss of activity.     

Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible and `locked'' substrates

Subtilisin BPN' hydrolysed N-acetyl-l-3-(2-naphthyl)-alanine methyl ester, N-acetyl-l-leucine methyl ester and N-acetyl-l-valine methyl ester, faster than alpha-chymotrypsin. Of eight ;locked' substrates tested, only methyl 5,6-benzindan-2-carboxylate was hydrolysed faster by subtilisin, whereas the other esters were better substrates for chymotrypsin. Compared with the values for chymotrypsin, the stereospecific ratios during the hydrolysis of the optically active locked substrates by subtilisin were decreased by one and two orders of magnitude for bi- and tri-cyclic substrates respectively. The polar groups adjacent to the alpha-carbon atom of locked substrates did not contribute significantly to the reactivity of the more active optical isomers, but had a detrimental effect on the less active antipodes during hydrolysis by both the enzymes. These studies show that the binding site of subtilisin BPN' is longer and broader than that of alpha-chymotrypsin.     

Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible substrates

A series of arylalkanoate esters and alpha-acetamidoarylalkanoate esters were tested as substrates for alpha-chymotrypsin and subtilisin BPN'. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of alpha-chymotrypsin is roughly 1.1nm (11A) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the alpha-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.     

Sugar Specificity in the Induction and on the Activity of Streptomyces α-d-Galactosidases

The α-d-galactosidases of six Streptomyces strains were examined on their inducer susceptibility, substate specificity, and inhibitor susceptibility. In all strains examined, α-d-galactosidase was induced by d-galactose, but neither by d-fucose nor by l-arabinose. α-d-Fucosidase activity was always induced accompanying with α-d-galactosedase activity. β-l-Arabinosidase activity, however, was never observed. These α-d-galactosidases were purified to electrophoretically pure degree by successive ammonium sulfate and ethanol precipitation, and ion exchange and gel filtration chromatography. The purified preparations from six strains were different from each other in their chromatographic behaviors and in some physical properties, but they all showed strong α-d-fucosidase activity as well. The α-d-galactosidase activities were strongly inhibited by d-galactose and l-arabinose, but scarcely by d-fucose. On the other hand, their α-d-fucosidase activities were inhibited by d-fucose as well as by d-galactose and l-arabinose.     

Substrate Specificity of Mammalian N-Terminal α-Amino Methyltransferase NRMT

N-Terminal methylation of free α-amino groups is a post-translational modification of proteins that was first described 30 years ago but has been studied very little. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono-, di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-amino-methylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products.