Stability of butyrylcholinesterase: thermal inactivation in water and deuterium oxide

Irreversible thermal inactivation of the tetrameric form of human plasma butyrylcholinesterase (cholinesterase; EC 3.1.1.8) was studied in water and in deuterium oxide at pH 7 in the temperature range 53-65 degrees C. The enzyme inactivation follows a complex kinetics that may be described by the sum of two apparent first-order processes. The Eyring plot for enzyme inactivation exhibits a wavelike discontinuity over a span of 2 C degrees around 58 degrees C. This transition was interpreted in terms of equilibrium between two temperature-dependent conformational states. Though 2H2O does not alter the overall multistep inactivation process, a slight solvent isotope effect was observed: a stabilizing effect and a shift in the transition temperature. A comparison between several enzyme preparations revealed differences in thermodynamic activation parameters of inactivation suggesting microheterogeneity in enzyme structures. Kinetics of inactivation of usual (E1uE1u) and atypical (E1aEa1a++) enzymes were compared. The atypical enzyme was found to be more stable than the usual phenotype.     

Accelerated death kinetics of Aspergillus niger spores under high-pressure carbonation

The death kinetics of Aspergillus niger spores under high-pressure carbonation were investigated with respect to the concentration of dissolved CO2 (dCO2) and treatment temperature. All of the inactivation followed first-order death kinetics. The D value (decimal reduction time, or the time required for a 1-log-cycle reduction in the microbial population) in the saline carbonated at 10 MPa was 0.16 min at 52 degrees C. The log D values were linearly related to the treatment temperature and the concentration of dCO2, but a significant interaction was observed between them.     

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

The influence of dissolved CO(2) concentration on the death kinetics of Saccharomyces cerevisiae

AIMS: The effects of temperature and concentration of dissolved CO(2) on the inactivation of Saccharomyces cerevisiae were investigated using a plug-flow system. METHODS AND RESULTS: Several combinations of pressure (4, 6, 8, 10 mega-Pa (MPa)) and temperature (30, 34, 36, 38 degrees C) were used. The D-values obtained were 0.14 min at 8 MPa and 38 degrees C, and 0.15 min at 10 MPa and 36 degrees C. The log D-values were related linearly to the treatment temperature and to the dissolved CO(2) concentration. The thermal resistance constant (zCO(2)(T)) was 9.5 degrees C in the media, including significant levels of CO(2), and the CO(2) resistance constant was z(temp.)(gamma)=7.2 gamma. CONCLUSION: This work has shown that inactivation followed first-order death kinetics, and the effects of temperature and CO(2) concentration were consistent through the critical temperature and pressure of CO(2). Therefore, it is feasible to estimate D-values at any temperature and any CO(2) concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: Non-thermal inactivation of micro-organisms in acidic beverages could be realized by the present technique.     

Molecular asymmetry in alkaline phosphatase of Escherichia coli

Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes. In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively. Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme. Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated. The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties. Structural implications of these results have been discussed.     

NADP(+)-malic enzyme from sugarcane leaves: structural properties studied by thermal inactivation

The irreversible thermal inactivation of the sugarcane leaf NADP(+)-malic enzyme was studied at 50 degrees C and pH 7.0 and 8.0. Depending on the preincubation conditions, thermal inactivation followed mono- or biphasic first-order kinetics. A two-step behavior in the irreversible denaturation process was found when protein concentration was sufficiently low. The protein concentration necessary to obtain monlphasic thermal inactivation kinetics was lower at pH 8.0 than at pH 7.0. The results suggest that biphasic inactivation kinetics are the consequence of the existence of two different oligomeric forms of the enzyme (dimer and tetramer), with the dimer being more stable in regards to thermal inactivation. The effects of the substrate and essential cofactors on the thermostability and equilibrium between the dimeric and tetrameric enzyme forms were also studied. Depending on the pH, NADP+, L-malate, and Mg2+ all had a protective effect on the stability of the dimeric and tetrameric species during thermal treatment. However, these ligands showed different effects on the aggregation state of the enzyme. NADP+ and L-malate induced dissociation, especially at pH 8.0, whereas Mg2+ induced aggregation of the protein. By studying the thermal inactivation kinetics at 50 degrees C and different pH values it was observed that the equilibrium between dimers and tetramers was dramatically affected in the range of pH 7.0-8.0. These results suggest that an amino acid residue(s) in the protein with an apparent pKa value of 7.7 needs to be deprotonated to stabilize aggregation of the enzyme to the tetrameric form.     

Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase and their role on hydrolysis of naringin solutions

The kinetics of thermal inactivation of A. terreus alpha-rhamnosidase was studied using the substrate p-nitrophenyl alpha-L-rhamnoside between 50 degrees C and 70 degrees C. Up to 60 degrees C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60 degrees C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60 degrees C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.     

APS kinase from Penicillium chrysogenum. Dissociation and reassociation of subunits as the basis of the reversible heat inactivation

Adenosine-5'-phosphosulfate (APS) kinase from Penicillium chrysogenum, loses catalytic activity at temperatures greater than approximately 40 degrees C. When the heat-inactivated enzyme is cooled to 30 degrees C or lower, activity is regained in a time-dependent process. At an intermediary temperature (e.g. 36 degrees C) an equilibrium between active and inactive forms can be demonstrated. APS kinase from P. chrysogenum is a dimer (Mr = 57,000-60,000) composed of two apparently identical subunits. Three lines of evidence suggest that the reversible inactivation is a result of subunit dissociation and reassociation. (a) Inactivation is a first-order process. The half-time for inactivation at a given temperature is independent of the original enzyme concentration. Reactivation follows second-order kinetics. The half-time for reactivation is inversely proportional to the original enzyme concentration. (b) The equilibrium active/inactive ratio at 36 degrees C increases as the total initial enzyme concentration is increased. However, Keq,app at 5 mM MgATP and 36 degrees C calculated as [inactive sites]2/0.5 [active sites] is near-constant at about 1.7 X 10(-8) M over a 10-fold concentration range of enzyme. (c) At 46 degrees C, the inactive P. chrysogenum enzyme (assayed after reactivation) elutes from a calibrated gel filtration column at a position corresponding to Mr = 33,000. Substrates and products of the APS kinase reaction had no detectable effect on the rate of inactivation. However, MgATP and MgADP markedly stimulated the reactivation process (kapp = 3 X 10(5) M-1 X s-1 at 30 degrees C and 10 mM MgATP). The kapp for reactivation was a nearly linear function of MgATP up to about 20 mM suggesting that the monomer has a very low affinity for the nucleotide compared to that of the native dimer. Keq,app at 36 degrees C increases as the MgATP concentration is increased. The inactivation rate constant increased as the pH was decreased but no pK alpha could be determined. The reactivation rate constant increased as the pH was increased. An apparent pK alpha of 6.4 was estimated.     

N-bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11

Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa inact of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue.     

Inactivation of the human thyroid peroxidase by ultrasound cavitation]

The inactivation kinetics of a human thyroid peroxidase protein fraction upon sonication (ultrasound frequency 27 kHz, power 60 W/cm2) of the enzyme solution in 15 mM phosphate buffer, pH 7.5, was studied. To quantitatively characterize the dependence of the slowest stage of the human thyroid peroxidase inactivation on temperature (36.0-50.4) degrees C, an effective constant of ultrasound inactivation rate Kin(US) was used. From the temperature dependence of Kin(US) at temperatures below 43 degrees C, the activation energy was estimated to be 8.11 kcal/mol. It was shown that the rate of human thyroid peroxidase inactivation strongly depends on the concentration of total protein in solution: the kin(US) value decreases more than sixfold in the protein concentration range from 0.2 to 0.8 mg/ml. It was also shown that poly(2-aminodisulfide-4-nitrophenol), its complexes with human serum albumin as well as the complexes human serum albumin--poly(gallic acid disulfide) substantially inhibit the ultrasound-induced inactivation of the enzyme and can be its effective stabilizers in the ultrasound cavitation field. This confirms the suggestion that active free radicals HO., O2.- and HO2. play a key role in the inactivation of human thyroid peroxidase. A general scheme of the inactivation of human thyroid peroxidase is proposed, which represents a chain of successive and parallel reversible and irreversible elementary steps.     

Inactivation of liver S-adenosylhomocysteine hydrolase in vitro of rats treated with erythro-9-(2-hydroxynon-3-yl)adenine.

S-Adenosylhomocysteine hydrolase activity decreased in vitro time-dependently in liver homogenates obtained from rats treated in vivo with erythro-9-(2-hydroxynon-3-yl)adenine, a potent inhibitor of adenosine deaminase. The inhibitor in itself had no effect on the stability of the hydrolase. The inactivation of S-adenosylhomocysteine hydrolase was irreversible, proceeded fairly rapidly at a low temperature (0 degrees C) and showed first-order reaction kinetics. Adenosine was found to accumulate in these tissue homogenates during storage. Several lines of evidence suggest that adenosine caused the observed suicide-like inactivation post mortem. Pre-incubation of purified S-adenosylhomocysteine hydrolase at 0 degrees C with adenosine showed a half-maximal inactivation rate at 33 microM substrate concentration; the rate constant of inactivation was 0.01 min-1. Inactivation during tissue preparation and storage complicates the assay of S-adenosylhomocysteine hydrolase activity in samples that contain an inhibitor of adenosine deaminase. These results also suggest that the decrease of S-adenosylhomocysteine hydrolase activity reported to occur in several disturbances of purine metabolism should be re-examined to exclude the possibility of inactivation of the enzyme in vitro.     

A second, slower inactivation process in acetylcholine receptor-rich membrane vesicles prepared from Electrophorus electricus

An agonist such as carbamylcholine or phenyltrimethylammonium induced a second, slower complete inactivation of acetylcholine receptor prepared from Electrophorus electricus. The rate of this inactivation of the receptor followed first-order kinetics. The rate constant of the inactivation increased with the agonist concentration until it reached a plateau, the value of which was 0.19 h-1 at 4.5 degrees C. The reaction was also temperature dependent, and the activation energy of the inactivation caused by 1 mM carbamylcholine was estimated to be 7.6 kcal/mol. The inactive receptor was reconverted to the active form with a rate constant of about 0.015 h-1 at 4.5 degrees C when the carbamylcholine concentration (0.1 mM) was reduced by 15-fold dilution in eel Ringer's solution. These results can be interpreted by adding, to the minimal reaction scheme proposed by the Hess group, a second, slower, reversible inactivation process either through the intact form or through the first desensitized form of the receptor binding two agonist molecules.     

A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase

A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.     

Inactivation of glucose-6-phosphate dehydrogenase in solution by low- and high-frequency ultrasound

Kinetics of inactivation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in 0.1 M phosphate buffer (pH 7.4) within temperature range from 36 to 50 degrees C was studied comparatively under conditions of exposure of enzyme solution to low-frequency (LF, 27 kHz, 60 W/cm2) or high-frequency (HF, 880 kHz, 1.0 W/cm2) ultrasound (USD). Inactivation of G6PDH was characterized by effective first-order rate constants: (k(in)) total (summarized) inactivation; (k(in)*) thermal inactivation; and (k(in)(usd)) ultrasonic inactivation. Dilution of enzyme solution from 20 to 3 nM was accompanied by a significant increase in the values of the three rate constants. The following inequality was valid in all cases: k(in) > k(in)*. The rate constants increased upon increasing the temperature. The Arrhenius plots of the temperature dependencies of k(in) and k(in) (usd) have a salient point at 44 degrees C. The activation energy (Eact) of the total inactivation of G6PDH was higher than Eact for the process of ultrasonic inactivation of this enzyme. The two values were found to depend on USD frequency: Eact in case of inactivation with low-frequency ultrasound (LF-USD) was higher than in case of inactivation with high-frequency ultrasound (HF-USD). The rate of the ultrasonic induced inactivation of this enzyme substantially decreased in the presence of low concentrations of traps of radicals HO. (dimethylformamide, ethanol, and mannitol). This fact supports the conclusion that free radicals are involved in the mechanism of the G6PDH inactivation in solutions exposed to LF-USD and HF-USD. Ethanol was an effective protector of G6PDH inactivation in enzyme solutions exposed to USD.     

Single-step unimolecular non-first-order enzyme deactivation kinetics

A two-parameter deactivation model is proposed to describe the kinetics of activity stabilization for some enzymes. The single-step unimolecular mechanism exhibits non-first-order deactivation kinetics since the final enzyme state, E(1) is not completely inactivated. The usefulness of the model is demonstrated by applying it to the inactivation of different enzymes. The influence of the concentration of active ester, ionic strength, and pH on the model parameters is examined during the inactivation of electric eel acetylcholinesterase.(25) In general, inactivators would decrease the level of activity stabilization, alpha(1), and increase the first-order inactivation rate constant, k(1). The effect of protecting agents would be to increase alpha(1) and to decrease k(1).     

Reaction conditions for laccase catalyzed polymerization of catechol

Poly(catechol) was synthesized in batch runs with laccase from Trametes versicolor (ATCC 200801). The polymerization reaction was conducted in a closed, temperature controlled system containing acetone and sodium acetate buffer for pH control. The effects of the solvent mixture, monomer (catechol), enzyme, medium pH and temperature on the polymerization rate were investigated with respect to initial reaction conditions and depletion rate of dissolved oxygen in the medium. Maximum initial reaction rate was attained with 10% (v/v) acetone-sodium acetate buffer at pH 5.0, 25 degrees C, 0.02 U/ml enzyme and 250 mg/l initial catechol and 10 mg/l dissolved oxygen. A general saturation enzyme kinetics response was observed for catechol substrate. Temperature rise supported the rate increase up to 45 degrees C, after which the rate tended to be stable due to a drop in dissolved oxygen concentration as well as enzyme instability.     

Enzyme deactivation

Enzyme deactivation kinetics is often first-order. Different examples of first-order deactivation kinetics exhibited by different enzymes under a wide variety of conditions are presented. Examples of both soluble and immobilized enzymes are presented. The influence of different parameters, chemical modification of specific residues, inhibitors, inactivators, protecting agents, induced conformational changes by external agents, enzyme concentration, and different substrates on the first-order inactivation kinetics of different enzymes is analyzed. The different examples presented from a variety of different areas provides a judicious framework and collection demonstrating the wide applicability of first-order deactivation kinetics. Examples of reversible first-order deactivation kinetics and deactivation-disguise kinetics are also presented.Different mechanisms are also presented to model complex enzyme deactivations. The non-series type mechanisms are emphasized and these involve the substrate and chemical modifiers. Substrate-dependent deactivation rate expressions that are of "separable" and "non-separable" type are presented. Rate expressions involving time-dependent rate constants along with their corresponding mechanisms are presented. Examples of enzymes that exhibit a deactivation-free grace period are also given. An interesting case of enzyme inactivation is the loss of activity in the presence of an auto-decaying reagent. The method is presented by which the intrinsic inactivation rate constants may be obtained. Examples of pH-dependent enzyme inactivation are presented that may be modelled by a five-step (or a simplified two-step) mechanism, and also by a single-step mechanism involving residual activity for the final state. Appropriate examples of enzyme inactivation are presented in each case to highlight the different mechanisms involved.     

Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn²⁺ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

Catalytic and immunochemical properties of ferritin conjugates with horseradish peroxidase

The human spleen ferritin--horseradish peroxidase conjugate (HRP--Fer) was synthesized by periodate oxidation of the enzyme carbohydrate fragment. The protein fraction containing 1-2 peroxidase molecules and characterized by kinetic homogeneity was obtained in the peroxidatic ortho-dianisidine (o-DA) oxidation reaction. Gel diffusion precipitation of HRP--Fer with peroxidases and ferritin antibodies was carried out. The precipitation confirms the retention by peroxidase and ferritin of their antigenic properties. The kinetics of peroxidatic oxidation of o-DA by the HRP--Fer conjugate was studied within the temperature interval of 15-37 degrees C. The value of catalytic constant for this reaction exceeds that for native peroxidase 1.75-fold. A kinetic analysis of thermal inactivation of peroxidase and its conjugate was performed within the temperature range of 40-65 degrees C. The effective rate constants of inactivation obtained from the first order equation are higher for HRP--Fer than for the native enzyme. The effect of pH on the rates of inactivation of HRP--Fer and the non-modified enzyme was studied at 50 degrees C. The enzyme and its conjugate were shown to stabilize in acid media. The HRP--Fer conjugate can be used as an effective tool in immunoenzymatic assays of ferritin.     

Studies on the thermal inactivation of immobilized enzymes

The thermal inactivation of a great number of immobilized enzymes shows a biphasic kinetics, which distinctly differs from the first-order inactivation kinetics of the corresponding soluble enzymes. As shown for alpha-amylase, chymotrypsin, and trypsin covalently bound to silica, polystyrene, or polyacrylamide, the dependence of the remaining activities on the heating time can be well described by the sum of two exponential terms. To interpret this mathematical model function, the catalytic properties of immobilized enzymes (number of active sites in silica-bound trypsin, K(M) and E(a) values in silica-bound alpha-amylase and chymotrypsin) at different stages of inactivation and the influence of various factors (coupling conditions, addition of denaturants or stabilizers, etc.) on the thermal inactivation of silica-bound alpha-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound beta-amylase were compared by electron spin resonance (ESR) studies. The results suggest that the biphasic inactivation kinetics reflects two different pathways according to which catalytically identical enzyme molecules are predominantly inactivated.