Chemical modification of epsilon-amino lysine groups in horseradish peroxidase. Its effect on catalytic properties and spatial structure of the enzyme]

Effect of chemical modification of horseradish peroxidase lysine epsilon-amino groups by propionic, butyric, valeric, succinic anhydrides and trinitrobenzolsulfonic acid (TNBS) on catalytic properties of the enzyme is investigated. All the preparations of modified peroxidase have 100% peroxidase activity for o-dianizidine at pH 7.0, which indicates the absence of lysine epsilon-amino group in the enzyme active site. pH-dependencies of modified peroxidase relative activity are studied; modification by anhydrides of monobasic acids is not found to result in changes of the relative activity pH-profile, while modification by succinic anhydride widens it. Absorption and circular dichoism spectra of native and modified peroxidase within 260--270 nm are obtained, some changes in the enzyme tertiary structure after its epsilon-amino groups modification are observed. Modification of four epsilon-amino groups by buturic and succinic anhydrides and of three epsilon-amino groups by TNBS is found to increase the regidity of protein surrounding of heme, and modification of six epsilon-amino groups by TNBS results in more unwrapped enzyme structure as compared with its native molecule.     

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.     

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.     

The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits. Kinetic and thermodynamic aspects.

Formyltetrahydrofolate synthetase monomers are converted to catalytically active tetramers in the presence of monovalent cations. The stoichiometry of the reaction is 4M + 2C+ in equilibrium M4C2(2+). A positive deltaS compensates for an unfavorable positive deltaH so that the overall reaction is exergonic. Both deltaH and deltaS become more positive as the temperature is increased. Association of subunits of the enzyme prepared from Clostridium cylindrosporum is second order with respect to monomer concentration, consistent with a rate-determining dimerization step. Activation parameters for this step at 20 degrees are: deltaG, 12.6 kcal mol-1; deltaH, 12.5 kcal mol-1; deltaS, -05 e.u. The rate-limiting step for the cation-dependent association of Clostridium acidi-urici monomers is believed to be a conformational alteration since first order kinetics is observed. The Eyring plot of the kinetic data obtained for the C. acidi-urici system has a sharp break at 15 degrees. Activation parameters for cation-induced association at 20 degrees are: deltaG, 21.5 kcal mol-1; deltaH, 14.0 kcal mol-1; deltaS, -26.6 e.u.     

Protein stabilization via hydrophilization. Covalent modification of trypsin and alpha-chymotrypsin

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. Alpha-chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of alpha-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 degrees C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.     

Peroxidase activity of succinylated catalase

The catalase succinylation by succinic anhydride excess results in an almost complete dissociation of the enzyme into subunits possessing no catalase activity. The catalase subunits show the peroxidatic activity on o-dianisidine oxidation. The oxidation kinetics of this substrate by the succinylated enzyme was studied at various temperatures. The activation energy for this process is 10.1 kcal/mole. Within the temperature range of 31-65.5 degrees, the succinylated enzyme thermostability was studied by monitoring the peroxidatic activity decrease upon o-dianisidine oxidation. The activation energy for the succinylated catalase thermoinactivation equals to 15.5 kcal/mole. The peroxidatic activity of catalase subunits obtained by enzyme succinylation and acidic solution treatment was compared to that of horseradish peroxidase in the oxidation of the same substrate, i.e., o-dianisidine.     

Thermostability at ultrahigh temperatures of thermolysin and a protease from a psychrotrophic Pseudomonas.

Thermal inactivation at 110-150 degrees C of thermolysin (EC 3.4.24.4), produced by the thermophile Bacillus thermoproteolyticus, and the extracellular protease of Pseudomonas sp. MC60 a psychotroph, were investigated at 130 degrees C, both enzymes had approximately the same deltaH (22 kcal/mol) and deltaS (-13.5 cal/mol per degree) values. Both enzymes contain zinc and calcium. The amino acid compositions of the enzymes were similar except that MC60 protease exhibited a more typical tyrosine content. Comparable heat resistance at extreme temperatures of enzyme produced by psychrotrophic and thermophilic organisms emphasizes the difference between molecular properties that resist denaturation at elevated temperatures and those that allow reversible denaturation.     

Microcalorimetric determination of thermochemical parameters of the phosphofructokinase reaction]

A calorimetric procedure for determining deltaH, deltaG, deltaS and Keq of a bimolecular reaction with two or more products is described. By using this method the thermodynamic parameters of the phosphofructokinase reaction are determined. At pH 7.0 and 25 degrees C a reaction enthalpy of-6.96kcal/mole was found after correction for the neutralization enthalpy of the buffer and of the enthalpy difference of the magnesium complexes of ATP and ADP, respectively. The free energy of the phosphofructokinase reaction has been found under these conditions to be -3.96kcal/mole.     

Gel filtration studies on oxyhemerythrin. II. Effect of temperature and ionic strength on the association-dissociation equilibria.

The effects of temperature and ionic strength on the association of oxyhemerythrin have been studied. deltaH degrees and deltaS degrees for association at pH 7.0 are -2.6 kcal and +16.5 eu per mol of monomer. These values suggest that solvent adjacent to the surface of the protein undergoes rearrangement on association. Increasing ionic strength is observed to promote dissociation while decreasing the rate of attainment of equilibrium between monomers and octamers. Qualitatively similar results are observed on lowering the pH from 7.0 to 4.8, thereby linking the effects of increasing ionic strength to those of protonation of specific amino acid residues at the subunit contacts of hemerythrin. The apparent enthalpy of ionization of the amino acid residue controlling dissociation at acidic pH was found to be -1.9 to +2.1 kcal/mol. These values are consistent with a carboxyl group.     

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.     

Catalytic and thermodynamic properties of the urocanate hydratase reaction.

Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.     

Kinetics of N-dealkylation of amines with participation of microsomal cytochrome P-450]

Within the temperature range of 20-37 degrees C the kinetics of the demethylation reactions of a variety of amines with participation of hepatic microsomal cytochrome P-450, NADPH and O2 has been studied. Catalytical rate constants for all the substrates have been determined in generalized forms. Activation parameters deltaH* and deltaS* are also estimated. There is a linear relationship between deltaH* and deltaS*: deltaH*=20.7 kcal+366 degrees K deltaS*. Compensation relationship is characterized by a coefficient alpha=366 degrees/Taverage=1.21. The nature of the limiting step of the enzymatic amine demethylation involving cytochrome P-450 of hepatic microsomes is discussed.     

Kinetic and spectral parameters of the amines oxidative N-dealkylation with participation of the liver microsomal cytochrome P-450. Amines oxidation with organic hydroperoxides

Kinetics of demethylation of a number of amines involving hepatic microsomal cytochrome P-450 and organic hydroperoxides (tret-butyl- and cumylhydroperoxide) have been investigated. Decomposition rate constants for the substrate-cytochrome P-450-ROOH complexes have been determined in a generalized form. Activation parameters, deltaH* and deltaS*, are calculated for decomposition of the complexes. There is a linear relation between deltaH* and deltaS*: deltaH*=18.7 kcal + 333 degrees K deltaS*. Compensation relationship is characterized by the value of alpha=333 degrees K/Taverage=1.11. The nature of the limiting step in the cytochrome P-450-NADPH-O2-system and the cytochrome P-450-ROOH-system is discussed.     

Lys70 of E. coli quinolinate phosphoribosyltransferase is protected from chemical modification by formation of an inhibitor complex

Quinolinate phosphoribosyltransferase was examined for susceptibility to different chemical modification reagents. Loss of enzyme activity with trinitrobenzenesulfonate (TNBS) occurred when 1.1 lysines per subunit were modified. Tryptic digestion of the modified enzyme followed by HPLC-MS analysis of the peptides showed Lys70 reacts with TNBS. Based on x-ray studies, this amino acid participates in a conformational change distant from the active site.     

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.     

Stacking interactions of nucleobases: NMR-investigations. I. Self association of N6, N9-dimethyladenine and N6-dimethyl-N9-ethyladenine.

The selfassociation of N6,N9-dimethyladenine and N6-dimethyl-N9-ethyladenine has been studied by means of NMR technique. The thermodynamic quantities have been calculated using an isodesmic NMR model with three NMR parameters (the monomer shift deltaM and two complex shifts delta2 and delta3). The dependence of the thermodynamic quantities on the NMR parameters is discussed. Special attention is given to the determination of deltaM and its temperature dependence. Calculations with delta3 = 2 - delta2 and deltaM taken independently of temperature result in an average entropy deltaS = - 17.9 +/- 1.8 e.u. for N6,N9-dimethyladenine and deltaS = - 16.7 +/- 1.7 e.u. for N6-dimethyl-N9-ethyladenine and in an average enthalpy deltaH = - 7.2 +/- 0.6 kcal - mol-1 for both substances investigated.     

Kinetics of the oxidation of Rhus vernicifera stellacyanin by the Co(EDTA)-- ion.

A kinetic study of the oxidation of the copper(I) form of the blue copper protein stellacyanin (St(I) by Co(EDTA)-- has been performed. Observed rate constants approach a saturation limit with increasing [Co(EDTA)--] at pH 7, consistent with a mechanism involving rapid pre-equilibrium oxidant-protein complex formation followed by rate-limiting intramolecular Cu(I) to Co(III) electron transfer: Co(EDTA)-- + St(i Qp in equilibrium Co(EDTA)-- ---St(I) Co(EDTA)-- ---St(I) k2 leads to Co(EDTA)2-- ---St(II) (Qp = 149 M--1, k2 = 0.169 sec--1; 25.1 degrees, pH 7.0 mu 0.5 M (phosphate)). Activation parameters based on k2 (deltaH not equal to = 1.8 kcal/mol, deltaS not equal to = --56 cal/mol-deg) indicate that the electron transfer process is substantially nondiabatic, in marked contrast with results obtained for Co(phen) 3 3+ as the oxidant. Linear kobsd VS. [Co(EDTA)--] plots are reported for the Co(EDTA)-- oxidation of cuprous stellacyanin at pH 10 (k = 8.9 M--1 sec--1; 25.0, pH 10, mu 0.5 M (carbonate); DELTaH not equal to 11.3 kcal/mol, deltaS not equal to = -16 cal/mol-deg) and at pH 7 in the presence of excess EDTA (k = 21.2 M--1 sec--1; 25.1 degree, pH 7.0, mu 0.5 M (phosphate), [EDTA] tot = 5 X 10(--4) M; deltaH not equal to = 5.9 kcal/mol, delta S not equal to = --33 cal/mol-deg). It is concluded that Co(EDTA)-- adopts an electron transfer mechanism similar to that preferred by Co(phen)33+ under conditions where the oxidant is prevented from binding strongly to reduced stellacyanin.     

Studies on mammalian intestinal peroxidase.

A peroxidase, purified from rat small intestine to apparent homogeneity as judged by polyacrylamide-gel electrophoresis, exhibited an absorbance ratio (A412/A280) of 0.783. Its Mr (44000 +/- 1000) and spectral properties were similar to those of the pig intestinal enzyme. The velocity constant for the reaction between rat intestinal peroxidase and hydrogen peroxide was found to be 1.8 x 10(7) M-1 . s-1. Benzhydroxamic acid inhibited the peroxidative oxidation of guaiacol by intestinal peroxidase from both species but the concentration required to cause half-inhibition of the enzyme from the rat was higher by one order of magnitude than for the pig enzyme. The amino acid composition of highly-purified pig intestinal peroxidase showed a relative abundance of basic amino acids (lysine and arginine) and was similar to that of lactoperoxidase, but not that of myeloperoxidase. The initial ten amino acid residues of this enzyme (the first reported partial sequence for a mammalian peroxidase) were also determined.     

Intermolecular interaction of adenosine-5'-phosphate with phenylalanine and tryptophan by nuclear magnetic resonance]

Equilibrium constants (K) of the complex formation of adenosine-5'-phosphate with phenylalanine (27 l/mol) and tryptophan (67 l/mol) in water (D2O, pD 9.9) have been determined. The comparison of the equilibrium constant values allowed to find the sequence of probabilities of the approach of aromatic amino acids to adenosine-5'-phosphate (tryptophan greater than tyrosine greater than phenylalanine). It has been shown that the dependence of lg K on l/T in the system phenylalanine-adenosine-5'-phosphate is non-linear. The ranges of the positive values of enthalpy (deltaH) and entropy (deltaS) changes of the complex phenylalanine-adenosine-5'-phosphate formation in the temperature interval 28-65 degrees have been found (deltaH = 0.2-1.5 kcal/mol, delta S = 14-50 e.u.). The conclusion about the hydrophobic interaction of aromatic cycles of nucleotide and amino acids has been deduced from the received data.     

Phosphoenolpyruvate carboxylase of Escherichia coli. The role of lysyl residues in the catalytic and regulatory functions.

Phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of E. coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased. The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP.