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     

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.     

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.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

The interaction of α-chymotrypsin with isosteric substrates of different charge type

1. The synthesis of three substrates of alpha-chymotrypsin of closely similar steric requirements but different charge type is reported. 2. The interaction of these compounds [SS-dimethyl-(l-3-carboxymethyl-3-acetamido)propyl sulphonium iodide, l-2-acetamido-5-methylhexanoic acid methyl ester and N-acetyl-l-glutamic acid alpha-methyl ester] with alpha-chymotrypsin has been studied. 3. For the charged substrates, values of k(0) are two orders of magnitude smaller than, and values of K(m) two orders of magnitude larger than, the corresponding values for the uncharged isostere. 4. The results are interpreted in terms of the known specificity of the enzyme, and the relationship between binding and kinetic specificities is discussed.     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

An examination of the utility of photogenerated reagents by using α-chymotrypsin

As a test of the labelling characteristics of photogenerated reagents, an aryl azide was photolysed in the aromatic-binding locus of a protein of known tertiary structure. The acyl-enzyme derived from the reaction of alpha-chymotrypsin with the p-nitrophenyl ester of p-azido[(14)C]cinnamate was isolated and photolysed. About 60% of the acyl group is covalently bound to the protein after photolysis and deacylation, and labelled enzyme is inactive. The covalently attached label is localized in the C chain of chymotrypsin, and there are firm indications that the major labelled tryptic fragment of the C chain is that which constitutes the aromatic-binding locus of the enzyme. The high degree of labelling of that portion of the protein molecule predicted on the basis of the known chemistry and structure of alpha-chymotrypsin, provides gratifying confirmation of the utility of the photo-labelling method.     

Enhancement of mycobactericidal activity of glutaraldehyde with α,β-unsaturated and aromatic aldehydes

Summary Several ,-unsaturated and aromatic aldehydes were evaluated for antimicrobial activity usingMycobacterium bovis as the test strain. Activity of most of the compounds was determined in the presence and absence of 2% glutaraldehyde. Several compounds highly active against this organism, e.g. 2-pentenal, benzaldehyde, ando-phthalaldehyde showed rapid kill of >10⁵ CFU ml^–1 in 5 min. Activity of ,-unsaturated compounds substituted in the ₁ position showed increasing activity with increasing chain length. Of the aromatic aldehydes tested, benzaldehyde andp-dimethylamino benzaldehyde showed little activity alone, but when combined with 2% glutaraldehyde showed increased activity. Substituents added to the benzaldehyde ring (nitro, chloro, methyl, and methoxy) all detracted from the synergism, but still showed increased activity over the activity of 2% glutaraldehyde. The same affect was noted with disubstituted benzaldehyde compounds but not with substitutedo-phthaladehyde (2-formylformaldehyde).     

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.     

Exploring the thermal stability of α-chymotrypsin in protic ionic liquids

Ammonium based ionic liquids (ILs) are biocompatible co-solvents that stabilize the native state of proteins. Experimentally, we have explored the stability of α-chymotrypsin (CT) in the presence of nine ILs, i.e., diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), diethylammonium dihydrogen phosphate (DEAP), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), trimethylammonium acetate (TMAA), trimethylammonium hydrogen sulfate (TMAS), trimethylammonium dihydrogen phosphate (TMAP). Thermodynamic folding properties such as transition temperature (T_m), Gibbs free energy change of unfolding (ΔG_U), enthalpy change (ΔH) and heat capacity change (ΔC_p) of CT in ILs are obtained by fluorescence spectra analysis. Fluorescence and circular dichroism (CD) spectroscopy experiments were performed to probe CT stabilization and structural changes in the presence of ILs. Our experimental results suggest that the ILs act as stabilizers for the CT structure and the stability of CT depends on the structural arrangement of the ions of ILs. Our experimental results reveal that ILs (DEAA, DEAS and DEAP) having more hydrophobic ammonium cations [DEA⁺] are weak stabilizers for CT, while trimethyl ammonium cations [TMA⁺] ILs having small alkyl chain length such as TMAA, TMAS and TMAP are strong stabilizers and therefore more biocompatible for the native structure of CT.     

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.     

A comparison of the crosslinking abilities of glutaraldehyde,formaldehyde and α-hydroxyadipaldehyde with bovine serum albumin and casein

Summary The crosslinking effects of formaldehyde, -hydroxyadipaldehyde and glutaraldehyde have been compared by various techniques. Using a micro-Ouchterlony technique with an aldehyde treated bovine serum albumin-rabbit anti-bovine serum albumin system it was found that glutaraldehyde prevented precipitin line formation except at very high titres of antibody. The effects of formaldehyde and -hydroxyadipaldehyde were less marked. Cellulose acetate electrophoresis of aldehyde treated bovine serum albumin showed an increase in mobility compared with the untreated protein. Starch gel electrophoresis of aldehyde fixed liver slices showed no protein loss after glutaraldehyde fixation whereas the other aldehydes permitted proteins to be extracted. Polyacrylamide gel electrophoresis of the aldehyde treated bovine serum albumin showed a little change in mobility after formaldehyde and -hydroxyadipaldehyde treatment and a little polymer formation. Glutaraldehyde on the other hand produced much polymer. These findings were confirmed by gel filtration with Sephadex G-200. Intermolecular crosslinking with glutaraldehyde was dependant on the aldehyde concentration.     

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.     

Increase of catalytic activity of α-chymotrypsin in organic solvent by co-lyophilization with cyclodextrins

-Chymotrypsin was lyophilized in the presence of 2,3,6-tri-O-methyl -cyclodextrin, and it displayed activity 40 fold higher than free -chymotrypsin for transesterification in acetonitrile. -Chymotrypsin which was co-lyophilized with hydroxypropylated - or -cyclodextrins retained more than 98% of its initial activity after 6 h incubation in acetonitrile.     

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.     

Effect of the cross-linker on the general performance and temperature dependent behaviour of a molecularly imprinted polymer catalyst of a Diels–Alder reaction

Here we present a series of molecularly imprinted polymers capable of catalysing the Diels–Alder reaction between benzyl 1,3-butadienylcarbamate (1) and N,N-dimethyl acrylamide (2). The polymer systems studied here demonstrated an unusual cross-linker and temperature dependent behaviour, namely that polymer catalysis of the Diels–Alder reaction was lower at elevated temperature, in contrast to the solution reaction. Furthermore, not only was the catalytic activity significantly influenced by the choice of cross-linker, but in a similar fashion also the extent of the temperature effect, indicating a close relationship between catalysis and the observed inhibition. Molecular dynamics simulations of both the polymer systems studied were used to provide insight into the molecular background of transition state stabilisation, and differences in properties of the systems based on different cross-linkers.     

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.     

Rate-adjusted cross-linking reaction by photoresponsive α-bromoaldehyde (PBA)-conjugated ODN

We developed a photoresponsive α-bromoaldehyde-conjugated oligonucleotide (PBA-ODN). The PBA-ODN selectively reacted and formed covalent bonds with target oligonucleotides having adenine or cytosine at the frontal position of the aldehyde derivative. Kinetic studies revealed that PBA-ODN has increased kinetic rates for the formation of cross-linked duplexes compared with the corresponding α-chloroaldehyde-conjugated oligonucleotide (PCA-ODN).     

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.