Chemical modification of lysine epsilon-NH2-groups in horseradish peroxidase. Its effect on enzyme stability. Temperature dependence of thermo-inactivation constants for native and modified peroxidase]

Thermostability of horseradish peroxidase modified by acetic, propionic, butyric, valeric and succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) is studied within the temperature range of 56-80 degrees C. Acylation of 4 amino groups and arylation of 3 amino groups with TNBS are found to stabilize the enzyme, while modification of 6 groups decreases the enzyme stability. Chemical modification of peroxidase does not change its pH-dependence with respect to enzyme thermostability. Thermodynamic activation parameters of irreversible thermoinactivation are determined for native and modified peroxidase. Native peroxidase has deltaH not equal to = 30+/-1 kcal/mole and deltaS not equal to = 14 e. e.; modified by acid anhydrides peroxidase has deltaH not equal to within 64-87 kcal/mole and deltaS not equal to within 110-178 e. e. depending on the nature of a modifying agent. The effect of the structure of a radical introduced into the enzyme molecule, and of a number of modified epsilon-amino groups on thermoinactivation deltaH not equal to and deltaS not equal to values is discussed.     

Chemical modification of the epsilon-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme.

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.     

Specific modification of free lysine amino groups of histidine decarboxylase from Micrococcus sp. n. by trinitrobenzene sulfonic acid]

It has been found that 14 lysine residues are accessible for trinitrobenzene sulfonic acid (TNBS) in the molecule of histidine decarboxylase (HDC). The other 62 lysine residues in the molecule of native HDC are masked and inaccessible for TNBS. It is demonstrated that the SH- and alpha-amino groups of methionine are not modified by TNBS. A correlation between the decarboxylase activity of the enzyme and the degree of its trinitrophenylation has been studied. HDC, whose molecule contains 3--9 TNP groups, retains up to 90--97% of its initial activity. Trinitrophenylation of 14 lysine residues induces inactivation of HDC by 33--34%, which probably depends on conformational changes or steric hindrances, occurring in the catalytic site of the modified active centre of HDC. Using circular dichroism and fluorescence methods as well as disc-electrophoresis in polyacrylamide gel, it has been shown that trinitrophenylation does not cause any significant changes in the enzyme structure. The TNP groups have been found to be localized in the large and small subunits of the HDC molecule.     

The structure and function of ribonuclease T1. XXIII. Inactivation of ribonuclease T1 by reversible blocking of amino groups with cis-aconitic anhydride and related dicarboxylic acid anhydrides.

Ribonuclease T1 [EC 3.1.4.8] was inactivated rapidly by treatment at pH 8.0 and 0 degrees C with cis-aconitic anhydride and related dicabroxylic acid anhydrides, including citraconic, maleic, and succinic anhydrides. Under reaction conditions used, roughly 90% inactivation occurred within 30 min. Analyses of the inactivated enzymes indicated that the reaction took place fairly specifically at the alpha-amino group of the N-terminal alanine and the epsilon-amino group of lysine-41. Upon incubation of these inactivated enzymes at pH 3.6 and 37 degreeC, the activity was regenerated to various extents, depending on the nature of the introduced acyl groups. Under these conditions, the enzyme modified with cis-aconitc anhydride or citraconic anhydride recovered much of the origninal activity after 48 h whereas the enzyme modified with maleic anhydride recovered its activity only partially. Practically no activity was regenerated in the case of the enzyme modified with succinic anhydride under these conditions. The inactivation appears to be due mainly to the effect of the carboxyl group introduced at the epsilon-amino group of lysine-41. The results suggest the usefulness of cis-aconitic anhydride as a reversible blocking reagent for amino groups in proteins.     

Chemical modification of papain for use in alkaline medium

Chemical modification is a useful method to recognize and modify functional determinants of enzymes. Papain, an endolytic cysteine protease (EC3.4.22.2) from Carica papaya latex has been chemically modified using different dicarboxylic anhydrides of citraconic, phthalic, maleic and succinic acids. These anhydrides reacted with five to six amino groups of the lysine residues in the enzyme, thereby changing the net charge of the enzyme from positive to negative. The resultant enzyme had its optimum pH shifted from 7 to 9 and change in temperature optima from 60 to 80 °C. The modified papain also had a higher thermostability. Stability of the modified papain was further increased by immobilization of the enzyme either by adsorption onto inert matrix or by entrapment in polysaccharide polymeric gels. Entrapment in starch gel showed better retention of enzyme activity. Incorporation of modified and immobilized enzymes to branded domestic detergent powders was found to have very good activity retention. The papain entrapped in starch gel showed better stability and activity retention than in other carbohydrate polymers when added to domestic detergent powders.     

Effect of succinylation (3-carboxypropionylation) on the conformation and immunological activity of ovalbumin.

The epsilon-amino groups of ovalbumin were modified with succinic anhydride; as many as 16 lysine residues were succinylated (3-carboxypropionylated). The five succinylated derivatives thus prepared were homogeneous with respect to the extent of chemical modification as shown by electrophoretic and immunological data. Succinylation of the amino groups altered electrophoretic mobility and isoionic pH of ovalbumin in the expected direction. U.v.-absorption and fluorescence spectra suggested changes in the microenvironment of the chromophores in the modified proteins. The difference-spectral results showed greater exposure of tyrosine and tryptophan residues in the succinylated ovalbumin. Increase in susceptibility to tryptic digestion, Stokes radius and intrinsic viscosity of native ovalbumin, which was observed on successive increase in the chemical modification, demonstrated a conformational change that was proportional to the extent of modification. The loss of immunological reactivity caused by chemical modification also indicated a conformational change in succinylated ovalbumin. The fact that the intrinsic viscosity of maximally modified ovalbumin was less than one-third of that for the completely denatured protein in 6M-guanidinium chloride suggested that the modified protein contained significant residual native structure. The latter presumably accommodates some antigenic determinants accounting for 37% residual immunological activity observed with maximally succinylated ovalbumin.     

Reductive alkylation of lysine residues in subtilisin DY

Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.     

Chemical modification of lysine residues in Bacillus licheniformis alpha-amylase: conversion of an endo- to an exo-type enzyme

The lysine residues of Bacillus licheniformis alpha-amylase (BLA) were chemically modified using citraconic anhydride or succinic anhydride. Modification caused fundamental changes in the enzymes specificity, as indicated by a dramatic increase in maltosidase and a reduction in amylase activity. These changes in substrate specificity were found to coincide with a change in the cleavage pattern of the substrates and with a conversion of the native endo- form of the enzyme to a modified exo- form. Progressive increases in the productions of rho-nitrophenol or glucose, when para nitrophenyl-maltoheptaoside or soluble starch, respectively, was used as substrate, were observed upon modification. The described changes were affected by the size of incorporated modified reagent: citraconic anhydride was more effective than succinic anhydride. Reasons for the observed changes are discussed and reasons for the effectivenesses of chemical modifications for tailoring enzyme specificities are suggested.     

Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues.

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5'-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff's base complex between pyridoxal 5'-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5'-phosphate per mole of PGM dimer through the epsilon-amino group of a lysyl residue. The effect of pyridoxal 5'-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5'-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5'-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5'-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme. The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.     

Chemical modification of lysine residues at active-site of human placental estradiol 17 beta-dehydrogenase

Estradiol 17 beta-dehydrogenase (EC 1.1.1.62.) activity was decreased by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent for modification of epsilon-amino moiety of lysine residues in a protein. The inactivation exhibited pseudo-first-order kinetics, and was protected by oxidyzed cofactors. Stoichiometric studies showed that the complete inactivation was caused by modification of one lysine residue per molecule of the enzyme. Differential modification with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), TNBS and dithiothreitol (DTT) indicated that the residues of lysine and cysteine were located at the active-site and played an essential role in the catalytic function of the estradiol 17 beta-dehydrogenase.     

Identification of "buried" lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors.

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or "buried" amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is "buried" in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.     

Chemical modification of lysine residues of beta-1,3-glucanase from Spisula sachalinensis

The effects of chemical modification of the amino groups of lysine residues on the activity of beta-1.3-glucanase from Spisula sachalinensis were studied. Modification of two lysine residues per molecule did not affect either the enzyme activity with respect to laminarine, nor the Km value. The modified beta-1.3-glucanase retains the ability to catalyze the transglycosylation and cleaves the high molecular weight CM-pachyman at the same rate as does the native enzyme. No significant changes in the enzyme thermal stability were observed. Thus, the modified enzyme groups cannot be involved in the enzyme active center and are exposed on the surface of the protein globule. The chemical modification was shown to have no effect on the enzyme kinetics, which is essential for its immobilization.     

Physico-chemical properties of terrylytin modified by a copolymer based on vinylpyrrolidone

The proteolytic activity of terrylytin produced by the culture of Asp. terricola and modified by a water-soluble copolymer of vinylpyrrolidone and acrolein remained unchanged after enzyme modification. Using micro-thin layer chromatography, it was shown that the bulk of the epsilon-amino groups of lysine residues of the protein enter the reaction with the aldehyde groups of the polymeric matrix. The sedimentation and diffusion patterns of the polymerenzyme adduct demonstrated that the molecular weight of the modified enzyme is the total of molecular weights of its constituent components. Evidence from viscosimetry and gel chromatography allowed to develop a hydrodynamic model of the macromolecular product. It was shown that the rate of the enzyme inactivation in the solution calculated from the first order reaction equation depends on the nature of the enzyme electrochemical microenvironment. Under conditions close to physiological ones the rate inactivation constant for terrylytin modified by a neutral polymeric matrix is 10 times less than that for the native enzyme. At the isoelectric point (pH 4,6) a positively charged polymeric form of terrylytin is found to be the most stable one. The pH and temperature optima for casein hydrolysis remained unchanged throughout polymeric modification. The polymeric membrane did not hamper the diffusion during approximation of the substrates (casein and insulin) to the enzyme molecule during the catalytic act, which manifested itself in a constancy of Michaelis curves. Terrylytin modification by a copolymer causes an increase of stability with respect to trypsin proteolysis and a decrease of human blood plasma affinity for the inhibitors. The apparent inhibition constants for modified enzyme forms do not depend on the nature of electrochemical microenvironment and exceed that for native terrylytin 10-fold.     

Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives.

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.     

Low pKa lysine residues at the active site of sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is inactivated by iodoacetamide with the modification of two specific residues. Comparing the amino acid sequence and mass spectra of the peptide fragments containing the modified residues with those from the native enzyme, the modified residues were identified to be lysine. The pKa of these residues were estimated to be 8.5 and 6.7 from the pH dependence of inactivation in the presence and absence of the competitive inhibitor, acetate. These estimated pKa values are much lower than that of the epsilon-amino group of lysine residue. There may be unique microenvironments around these residues that activate their -amino groups to be susceptible to iodoacetamide. A possible role of the lysine residue with pKa 6.7 is discussed.     

Chemical modification studies on a lectin from winged-bean [Psophocarpus tetragonolobus (L.) DC] tubers.

The effect of chemical modification on a D(+)-galactose-specific lectin isolated from winged-bean tubers was investigated to identify the type of amino acid involved in its haemagglutinating activity. Various anhydrides of dicarboxylic acids, such as acetic anhydride, succinic anhydride, maleic anhydride and citraconic anhydride, modified 57-68% of the amino groups of the winged-bean tuber lectin. Treatment with N-acetylimidazole modified only 45% of the total amino groups. Reductive methylation of free amino groups modified 57% of the amino groups. Modification of the amino groups of the lectin by acetic anhydride and succinic anhydride did not lead to any significant change in the haemagglutinating activity (greater than or equal to 75% active). However, citraconylation and maleylation of the lectin led to a significant decrease in the haemagglutinating activity (less than or equal to 20% active). Acetylation and succinylation (3-carboxypropionylation) of the lectin led to a decrease in the pI value of the native lectin from approx. 9.5 to approx. 4.5. Treatment of the lectin with N-bromosuccinimide led to the modification of two and four tryptophan residues per molecule in the absence and in the presence of 8 M-urea respectively. The immunological identity of all the modified lectin preparations showed no gross structural changes except the lectin modified with N-bromosuccinimide in the presence of urea at pH 4.0.     

Effect of chemical modification on thermal stability of horseradish peroxidase]

Soluble preparations of horse radish peroxidase are obtained by means of its amino groups modification with glutaric aldehyde, maleic anhydride and inert proteins including albumin. The enzyme activity is found to decrease under the modification with glutaric aldehyde and to be unchanged at all other cases. Thermal stability of the enzyme preparations obtained is studied within the temperature range from 56 to 80 degrees C. Thermostability of glutaric aldehyde-modified peroxidase is approximately 2.5-fold decreased at 56 degrees C. Thermostability of other preparations exceeds the stability of native peroxidase in 25--90 times at 56 degrees C. Thermodynamic parameters of activation for the process of irreversible thermoinactivation of native and modified enzyme are calculated. A strong compensation effect between activation enthalpy and entropy values is observed, which were changed in 1.5--2 times, while the free activation energy is changed by 2--3 kcal/mol only. Possible mechanism of the change of the enzyme thermal stability under its chemical modification is discussed.     

Inhibition of pigeon liver fatty acid synthetase by specific modification of lysine residues with 2,4,6-trinitrobenzenesulphonic acid

Pigeon liver fatty acid synthetase was inactivated irreversibly by 2,4,6-trinitrobenzenesulphonic acid (TNBS). Biphasic inactivation of the enzyme was observed with the inhibitor. NADPH provided protection to the enzyme against inactivation by TNBS and the extent of protection increased with NADPH concentration indicating that the essential lysine residues are present at the NADPH binding site. The stoichiometric results with TNBS showed that 4 mol of lysine residues are modified per mole of fatty acid synthetase upon complete inactivation. The rapid reaction of two amino groups per enzyme molecule led to the loss of 60% of the enzyme activity. These approaches suggested that two lysine residues present at the active site are essential for the enzymatic activity of fatty acid synthetase.     

Chemical modification with functionalized ionic liquids: a novel method to improve the enzymatic properties of Candida rugosa lipase

Chemical modification of lysine residues in Candida rugosa lipase (CRL) was carried out using five different functional ionic liquids, and about 15.4–25.0 % of the primary amino groups of lysine were modified. Enzymatic properties of the native and modified CRLs were investigated in olive oil hydrolysis reaction. Improved thermal stability, catalytic activity in organic solvents, and adaptability to temperature and pH changes were achieved compared with the native enzyme. CRL modified by [choline][H₂PO₄] showed the best results, bearing a maximum improvement of 16.7 % in terms of relative activity, 5.2-fold increase in thermostability (after incubation at 45 °C for 5 h), and 2.3-fold increase in activity in strong polar organic solvent (80 % dimethyl sulfoxide) compared with the native enzyme. The results of ultraviolet, circular dichroism and fluorescence spectroscopy suggested that the change of the secondary and tertiary structures of CRL caused by the chemical modification resulted in the enhancement of enzymatic performance. The modification of CRL with functional ionic liquids was proved to be a novel and efficient method for improving the enzymatic properties of CRL.     

Chemical modification studies on alkaline phosphatase from pearl oyster (Pinctada fucata): a substrate reaction course analysis and involvement of essential arginine and lysine residues at the active site

Alkaline phosphatases (ALP, EC 3.1.3.1) are ubiquitous enzymes found in most species. ALP from a pearl oyster, Pinctada fucata (PALP), is presumably involved in nacreous biomineralization processes. Here, chemical modification was used to investigate the involvement of basic residues in the catalytic activity of PALP. The Tsou's plot analysis indicated that the inactivation of PALP by 2,4,6-trinitrobenzenesulfonic acid (TNBS) and phenylglyoxal (PG) is dependent upon modification of one essential lysine and one essential arginine residue, respectively. Substrate reaction course analysis showed that the TNBS and PG inactivation of PALP followed pseudo-first-order kinetics and the second-order inactivation constants for the enzyme with or without substrate binding were determined. It was found that binding substrate slowed the PG inactivation whereas had little effect on TNBS inactivation. Protection experiments showed that substrates and competitive inhibitors provided significant protection against PG inactivation, and the modified enzyme lost its ability to bind the specific affinity column. However, the TNBS-induced inactivation could not be prevented in presence of substrates or competitive inhibitors, and the modified enzyme retained the ability to bind the affinity column. In a conclusion, an arginine residue involved in substrate binding and a lysine residue involved in catalysis were present at the active site of PALP. This study will facilitate to illustrate the role ALP plays in pearl formation and the mechanism involved.