Conformational stability and dynamics of cytochrome <Emphasis Type="Italic">c</Emphasis> affect its alkaline isomerization

The alkaline isomerization of horse heart ferricytochrome c (cyt c) has been studied by electronic absorption spectroscopy in the presence of the Hofmeister series of anions: chloride, bromide, rhodanide and perchlorate. The anions significantly affect the apparent pK _a value of the transition in a concentration-dependent manner according to their position in the Hofmeister series. The Soret region of the absorption spectra is not affected by the presence of the salts and shows no significant structural perturbation of the heme crevice. In the presence of perchlorate and rhodanide anions, the cyanide exchange rate between the bulk solvent and the binding site is increased. These results imply higher flexibility of the protein structure in the presence of chaotropic salts. The thermal and isothermal denaturations monitored by differential scanning calorimetry and circular dichroism, respectively, showed a decrease in the conformational stability of cyt c in the presence of the chaotropic salts. A positive correlation between the stability, ΔG, of cyt c and the apparent pK _a values that characterize the alkaline transition indicates the presence of a thermodynamic linkage between these conformational transitions. In addition, the rate constant of the cyanide binding and the partial molar entropies of anions negatively correlate with the pK _a values. This indicates the important role of anion-induced solvent reorganization on the structural flexibility of cyt c in the alkaline transitions. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

How various substrates activate the process of enzymatic hydrolysis by different cholinesterases

Kinetic analysis of the activating effect of substrate on the cholinesterase catalysis is performed. There are determined values of coefficient of activation A in the pH zone 5 for the process of hydrolysis of acetylcholine, indophenylacetate (IPhA), and 2,6-dichlorophenolindoph enylacetate (DIPhA) by cholinesterase (ChE) of horse blood serum, as well as of IPhA and DIPhA by ChE of optical ganglia of the Pacific squid Todarodes pacificus. The phenomenon of activation has not been revealed at hydrolysis of phenylacetate by the horse blood serum ChE. The conclusion is made that the cause of the activating effect of substrate on the process of enzymatic hydrolysis by ChEs of different origin is the presence of the onium grouping in the structure of substrates.     

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent

Kinetic enhancement of organophosphate hydrolysis is a long-standing challenge in catalysis. For prophylactic treatment against organophosphate exposure, enzymatic hydrolysis needs to occur at high rates in the presence of low substrate concentrations and enzymatic activity should persist over days and weeks. Here, the conjugation of small DNA scaffolds was used to introduce substrate binding sites with micromolar affinity to VX, paraoxon, and methyl-parathion in close proximity to the enzyme phosphotriesterase (PTE). The result was a decrease in K_M and increase in the rate at low substrate concentrations. An optimized system for paraoxon hydrolysis decreased K_M by 11-fold, with a corresponding increase in second-order rate constant. The initial rates of VX and methyl-parathion hydrolysis were also increased by 3.1- and 6.7-fold, respectively. The designed scaffolds not only increased the local substrate concentration, but they also resulted in increased stability and PTE-DNA particle size tuning between 25 and ~150 nm. The scaffold engineering approach taken here is focused on altering the local chemical and physical microenvironment around the enzyme and is therefore compatible with active site engineering via combinatorial and computational approaches.     

Binding of the Recombinant Proteinase Inhibitor Eglin c,of the Soybean Bowman-Birk Proteinase Inhibitor and of its Chymotrypsin and Trypsin Inhibiting Fragments to Leu-Proteinase,the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves: Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of K_a (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ～6.9, in the free Leu-proteinase, to ～5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - K_a = 2.2 × 10¹¹ M^-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - K_a = 3.2 × 10¹⁰ M^-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - K_a = 1.1 × 10⁶ M^-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of K_a, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10^-3 M, suggesting that the upper limit of K_a is 1 × 10² M^-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region     

Indophenol Chromogenic Substrates of Cholinesterases of Different Origin

An analysis of influence of indophenol substrate structure on rate of their enzymatic hydrolysis under action of cholinesterases (ChE) of different animals is carried out for the first time. Study of indophenylacetate (IPhA) and a group of isomeric dichloroderivatives as substrates of erythrocyte acetylcholinesterase, serum butyrylcholinesterase, and ChE from optical ganglia of the Pacific squid Todarodes pacificus allowed us to reveal a role of steric and inductive effects of the substrates molecule in enzymatic catalysis, as well as differences in substrate specificity of the studied ChE. This comparative enzymologic aspect of the work was evident to a greater degree at studying hydrolysis of choline (acetylcholine, acetylthiocholine) and indophenol (IPhA, 2,6-dichloroindophenylacetate, 2,6-dichloro-3´-methyl indophenylacetate) esters under action of mammalian blood ChEs, ChE from hemolymph of the gastropod mollusc Neptunea, and also of ChE from the nervous tissue of different species of Pacific squids and of the cabbage root fly. Differences in values of the kinetic parameters characterizing sorption and catalytic stages of the hydrolysis process are revealed. Comparison of substrate properties of choline and indophenol esters enabled us to compare enzymes in terms of hydrophobic-hydrophilic interactions.     

Water as a competitive inhibitor of lipase-catalysed esterification in organic media

Summary Kinetic parameters were determined for esterification of dodecanol and decanoic acid in hexane catalysed by lipases from Rhizomucor miehei and Candida rugosa, after pre-equilibration to different values of thermodynamic water activity (a_w). V_m increases with increasing a_w, but so do the K_m values for both substrates. The effect on K_m for the alcohol probably represents competition between the first product and the second substrate, as expected for Ping-Pong kinetics. The rise in K_m for the acid probably reflects the displacement of water molecules during substrate binding.     

How the various substrates activate the process of enzymatic hydrolysis by different cholinesterases

Kinetic analysis of the activating effect of substrate on the cholinesterase catalysis is performed. There are determined values of coefficient of activation A in the pH zone 5.0-7.5 for the process of hydrolysis of acetylcholine, indophenylacetate (IPA), and 2,6-dichlorophenolindophenylacetate (DIPA) by cholinesterase (ChE) of horse blood serum, as well as of IPA and DIPA by ChE of optical ganglia of the Pacific squid Todarodes pacificus. The phenomenon of activation has not been revealed at hydrolysis of phenylacetate by the horse blood serum ChE. The conclusion is made that the cause of the activating effect of substrate on the process of enzymatic hydrolysis by ChEs of different origin is the presence of the onium grouping in the structure of substrates.     

A goose-type lysozyme from ostrich (Struthio camelus) egg white: multiple roles of His101 in its enzymatic reaction

A goose-type lysozyme from ostrich egg white (OEL) was produced by Escherichia coli expression system, and the role of His101 of OEL in the enzymatic reaction was investigated by NMR spectroscopy, thermal unfolding, and theoretical modeling of the enzymatic hydrolysis of hexa-N-acetylchitohexaose, (GlcNAc)₆. Although the binding of tri-N-acetylchitotriose, (GlcNAc)₃, to OEL perturbed several backbone resonances in the ¹H–¹⁵N HSQC spectrum, the chemical shift of the backbone resonance of His101 was not significantly affected. However, apparent pK_a values of His101 and Lys102 determined from the pH titration curves of the backbone chemical shifts were markedly shifted by (GlcNAc)₃ binding. Thermal unfolding experiments and modeling study of (GlcNAc)₆ hydrolysis using a His101-mutated OEL (H101A-OEL) revealed that the His101 mutation affected not only sugar residue affinities at subsites ?3 and ?2 but also the rate constant for bond cleavage. His101 appears to play multiple roles in the substrate binding and the catalytic reaction.     

Preliminary kinetic characterization of a copper amine oxidase from rat liver mitochondria matrix

Kinetic measurements of a novel copper-dependent amine oxidase, purified from rat liver mitochondria matrix, were carried out using various substrates in a large pH (5.6–10.2) and ionic strength range (5–200 mM), in order to study the docking of substrates to the enzyme and, as a consequence, to verify the physicochemical characteristics of the active site. Relatively small changes of V _max values (approx. 2.5-folds) over the substrates tested, suggest that the rate determining step of the catalysis is only slightly affected by amine chemical structure. In contrast, the strong change of K _M and k _c/K _M values (approx. two orders of magnitude) indicates electrostatic control of the docking process, since the changes of K _M and k _c/K _M values appear due to the presence of positively charged groups in the substrate molecules. These results suggest the presence in the enzyme active site of two negatively charged amino acid residues which seem to interact with positively charged groups of the substrate molecules. Analogies and differences with bovine serum amine oxidase are also described.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

The Kinetics of Hydrolysis of Animal Fat by Pancreatic Lipase

Hydrolysis of lipids from beef fat by pancreatic lipase was studied. The maximum release of free fatty acids was shown to occur at 40°C for the first 3 h of the experiment. After this, transetherification was predominant. The main kinetic parameters were the following: maximum hydrolysis rate, V = 1.25 ± 0.1 mg fat/ml min; Michaelis constant, K _M ^H = 100 ± 12 mg fat/ml; constant of substrate inhibition, K _S = 10.0 ± 0.8 mg fat/ml; equilibrium constant, K _P = 277 ± 170 mg fat/ml; and activation energy of beef fat hydrolysis by pancreatic lipase, E _a = 19.1 ± 1.1 kJ/mole. The kinetic method used could be applied to development of the method for biotransformation of poorly assimilated fats into more valuable products.     

Analysis of the ionization constants and heats of ionization of reduced and oxidized horse heart cytochrome c

Simultaneous curve fitting for the ionization parameters of oxidized and reduced horse heart cytochrome c in 0.15M KCl and 20°C yields values for the ionization constants (as pK′) and the heats of ionization (ΔH_i) which can reconstruct either the potentiometric or thermal titration curves. Reduced cytochrome c requires 8 sets of groups, whereas oxidized cytochrome c requires 10 sets of groups. The additional groups in the oxidized preparation appear to involve the ferriheme (pK′, 9.25; ΔH_i, 13.7 kcal/mol) and a tyrosine (pK′ ? 10.24) that is not present in the reduced form. The potentiometric and thermal difference curves (reduced – oxidized) involve the appearance of 17 kcal/mol centered at pH 9.7 and 5.8 kcal/mol centered at pH 4.9. The carboxyl groups in both species appear to be normal for the hydrogen-bonded form. Only one histidine has normal ionization properties (pK′, 6.7; ΔH_i, 7.5 kcal/mol), as do 17 of the lysine residues (pK′, 10.8; ΔH_i, 11.5 kcal/mol).     

The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

Binding of 4-methyl umbelliferyl-α-D-glucoPyranoside to Vicia faba lectin: Fluorescence-quenching studies

On binding toVicia faba lectin, the fluorescence of 4-methylumbelliferyl-α-D-glucoPyranoside was quantitatively quenched showing that the interaction of 4-methylumbelliferyl-α-D-glucoPyranoside took Place in a binding environment. The binding of the fluorescent sugar was saccharide sPecific as evidenced by the reversal of 4-methylumbelliferyl-α-D-glucoPyranoside fluorescence quenching by D-fructose. The association constant,K _a, values for the 4-methylumbelliferyl-α-D-glucoPyranoside was determined by comPetition study emPloying reversal of fluorescence quenching of 4-methylumbelliferyl-α-D-glucoPyranoside by D-fructose. TheK _a value obtained for D-fructose was 1.07 ±0.03 X 10⁴ M^-1 and for 4-methylumbelliferyl-α-D-glucoPyranoside was 1.60 ±0.05 X 10⁴ M^-1 at 15°C. TheK _a values of 2.51 ±0.06 X 10⁴M^-1, l.26 ±0.02 X 10⁴ M^-1 and 0.56 ±0.01 X 10⁴M^-1, resPectively at 10°, 20° and 30°C were obtained from the ChiPman equation. The relative fluorescence quenching, ΔF _a, at infinite concentration of the free saccharide sites ofVicia faba lectin [P′] was 93.5% at 30°C and the binding constant for 4-methylumbelliferyl-α-D-glucoPyranoside lectin interaction as derived by Yank and Hanaguchi equation was 0.63 ±0.01 X 10⁴M^-1.     

Steady-state kinetic analysis of human cholinesterases over wide concentration ranges of competing substrates

Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate.Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate.For both enzymes, progress curves for hydrolysis of PhA at very low concentration (?K_m) in the presence of increasing concentration of ATC showed that: a) the competing substrate and the reporter substrate are hydrolyzed at the same time, b) complete hydrolysis of PhA cannot be reached above 1 mM competing substrate. This likely results from accumulation of hydrolysis products (P) of competing substrate and/or accumulation of acetylated enzyme·P complex that inhibit hydrolysis of the reporter substrate.     

Production and activity of Thermomonospora curvata cellulases on protein-extracted lucerne fibers

Summary Protein-extracted lucerne fibre (PELF) was evaluated as a carbon/energy source for cellulase production by Thermomonospora curvata and as a substrate for enzymatic conversion to soluble sugars. In shake cultures grown at 53°C, pH 8, maximal cellulase (50 endoglucanase IU and 0.7 filter paper IU/ml) was attained at a PELF concentration of 1.3% (w/v) in a vitamin-mineral salts medium buffered with 0.05 M concentrations of phosphate and N-2-hydroxyethylpiperazine-N-2-ethanesulfonate. Multiple endoglucanase enzymes were produced. The major form had an apparent molecular weight of 22 000 and K _m values of 0.23% and 0.56% for carboxymethylcellulose and PELF respectively. NaOH treatment of PELF increased its susceptibility to enzymatic hydrolysis. During enzymatic hydrolysis of NaOH-treated PELF (60°C, pH 6.1, 24h) two-thirds of total fibre carbohydrate was solubilized as cellobiose, melibiose, cellopentaose, cellotetraose, xylose and glucose in descending order of concentration.     

Comparative Enzymologic Study of Catalytic Properties of Blood Serum Cholinesterase of the American Mink Mustela vison

Comparative enzymologic study of catalytic properties of cholinesterase (ChE) in blood serum of the American mink Mustela vison Schr. has revealed several peculiarities of this enzyme. First, using the method of substrate–inhibitor analysis, homogeneity of the ChE preparation has been established, i.e. only one ChE has been found in mink serum. Second, the rate of acetylcholine hydrolysis was higher than of thiocholine substrates, among which propionylthiocholine was hydrolyzed at the highest rate. Third, propionylthiocholine had the highest V/K _M value that reflects to a degree affinity of the substrate to enzyme. Fourth, the phenomenon of substrate inhibition, which is not inherent for mammalian serum cholinesterases, is revealed and kinetically analyzed. Fifth, study of inhibitory specificity has not revealed differences of the mink serum ChE from other serum ChE.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Substrate specificity of chlorophyllase

Apparent Km and V_max values were obtained for hydrolysis of methyl and ethyl chlorophyllides a, methyl and ethyl pheophorbide a, and 9-hydroxymethyl pheophorbide a by chlorophyllase from Ailanthus altissima. Analysis of substrate specificity data for chlorophyllase indicates that the presence of a 9-keto group and a methyl alcohol group esterified at the 7-position in chlorophyll derivatives results in maximum binding affinity for substrates. Data on maximum reaction rates indicate that the rate-controlling step of hydrolysis occurs after release of the alcohol from the ester. Probable high affinity chlorophyllase inhibitors can be predicted on the basis of these specificity studies.     

Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as an intrinsic probe to characterize virtually every chemical process. Nowadays, this technique is extensively applied to determine thermodynamic parameters of biomolecular binding equilibria. In addition, ITC has been demonstrated to be able of directly measuring kinetics and thermodynamic parameters (k_cat, K_M, ΔH) of enzymatic reactions, even though this application is still underexploited. As heat changes spontaneously occur during enzymatic catalysis, ITC does not require any modification or labeling of the system under analysis and can be performed in solution. Moreover, the method needs little amount of material. These properties make ITC an invaluable, powerful and unique tool to study enzyme kinetics in several applications, such as, for example, drug discovery.In this work an experimental ITC-based method to quantify kinetics and thermodynamics of enzymatic reactions is thoroughly described. This method is applied to determine k_cat and K_M of the enzymatic hydrolysis of urea by Canavalia ensiformis (jack bean) urease. Calculation of intrinsic molar enthalpy (ΔH_int) of the reaction is performed. The values thus obtained are consistent with previous data reported in literature, demonstrating the reliability of the methodology.