Chemical modification of arginine residues of porcine muscle acylphosphatase

Acylphosphatase (acylphosphate phosphohydrolase, EC 3.6.1.7) from porcine skeletal muscle is inactivated by phenylglyoxal following pseudo-first-order kinetics. The dependence of the apparent first-order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is also first order with respect to the reagent concentration. Among the competitive inhibitors for the enzyme examined, inorganic phosphate and ATP almost completely, and Cl- partially, protect the enzyme against the inactivation. The dissociation constants for inorganic phosphate and ATP determined from protection experiments by these inhibitors agree well with those from inhibition experiments by them. These results support the idea that the modification occurs at the phosphate-binding site. The amino-acid analysis reveals the lack of reaction at residues other than arginine. Circular dichroism spectra of the modified enzymes show that the inactivation seems not to be due to denaturation of the enzyme resulting from the modification of the non-essential arginine residues. The relationship between the loss of the enzyme activity and the number of arginine residues modified in the presence and absence of ATP shows that one arginine residue is possibly responsible for the inactivation of acylphosphatase.     

Identification of an essential tyrosine residue in nitroalkane oxidase by modification with tetranitromethane

The flavoprotein nitroalkane oxidase from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with production of nitrite and hydrogen peroxide. The enzyme is irreversibly inactivated by incubation with tetranitromethane, a tyrosine-directed reagent, at pH 7.3. The inactivation is time-dependent and shows first-order kinetics for two half-lives of inactivation. Further inactivation can be achieved upon a second addition of tetranitromethane. A saturation kinetic pattern is observed when the rate of inactivation is determined versus the concentration of tetranitromethane, indicating that a reversible enzyme-inhibitor complex is formed before irreversible inactivation occurs. Values of 0.096 +/- 0.013 min(-1) and 12.9 +/- 3.8 mM were determined for the first-order rate constant for inactivation and the dissociation constant for the reversibly formed complex, respectively. The competitive inhibitor valerate protects the enzyme from inactivation by tetranitromethane, suggesting an active-site-directed inactivation. The UV-visible absorbance spectrum of the inactivated enzyme is perturbed with respect to that of the native enzyme, suggesting that treatment with tetranitromethane resulted in nitration of the enzyme. Comparison of tryptic maps of nitroalkane oxidase treated with tetranitromethane in the presence and absence of valerate shows a single peptide differentially labeled in the inactivated enzyme. The spectral properties of the modified peptide are consistent with nitration of a tyrosine residue. The amino acid sequence of the nitrated peptide is L-L-N-E-V-M-C-(NO(2)-Y)-P-L-F-D-G-G-N-I-G-L-R. The possible role of this tyrosine in substrate binding is discussed.     

Thermally perturbed rhodanese can be protected from inactivation by self-association

A fluorescence-detected structural transition occurs in the enzyme rhodanese between 30–40°C that leads to inactivation and aggregation, which anomalously decrease with increasing protein concentration. Rhodanese at 8 µg/ml is inactivated at 40°C after 50 min of incubation, but it is protected as its concentration is raised, such that above 200 µg/ml, there is only slight inactivation for at least 70 min. Inactivation is increased by lauryl maltoside, or by low concentrations of 2-mercaptoethanol. The enzyme is protected by high concentrations of 2-mercaptoethanol or by the substrate, thiosulfate. The fluorescence of 1,8-anilinonaphthalene sulfonate reports the appearance of hydrophobic sites between 30–40°C. Light scattering kinetics at 40°C shows three phases: an initial lag, a relatively rapid increase, and then a more gradual increase. The light scattering decreases under several conditions: at increased protein concentration; at high concentrations of 2-mercaptoethanol; with lauryl maltoside; or with thiosulfate. Aggregated enzyme is inactive, although enzyme can inactivate without significant aggregation. Gluteraldehyde cross-linking shows that rhodanese can form dimers, and that higher molecular weight species are formed at 40°C but not at 23°;C. Precipitates formed at 40°C contain monomers with disulfide bonds, dimers, and multimers. We propose that thermally perturbed rhodanese has increased hydrophobic exposure, and it can either: (a) aggregate after a rate-limiting inactivation; or (b) reversibly dimerize and protect itself from inactivation and the formation of large aggregates.     

Unfolding and inactivation of fatty acid synthase from chicken liver during urea denaturation

The inactivation and conformational changes of the multifunctional fatty acid synthase (acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85) from chicken liver have been studied in urea solution. The results show that complete inactivation of the fatty acid synthase occurs before obvious conformational changes with regard to the overall, beta-ketoacyl reduction and acetoacetyl-CoA reduction reactions. Significant conformational changes indicated by the changes of the intrinsic fluorescence emission and the circular dichroism spectra occurred at higher urea concentrations. The kinetic rate constants for the two phase inactivation and unfolding reactions were measured and semilogarithmic plots of the activity versus time gave curves which could be resolved into two straight lines, indicating that both the inactivation and unfolding processes consisted of fast and slow phases as a first-order reaction. The results from Lineweaver-Burk plots indicated that urea is a competitive inhibitor for acetyl-CoA and malonyl-CoA, with K(m) increasing with increasing urea concentrations. However, urea is a noncompetitive inhibitor for NADPH, the substrate of the overall reaction and beta-ketoacyl reduction reaction, and acetylacetate, the substrate of the beta-ketoacyl reduction reaction. Activation by low concentrations of urea was observed although this activation was only temporarily induced in an early stage of inactivation. The aggregation phenomenon of the fatty acid synthase in a certain concentration range of urea (3-4 M) was also observed during unfolding. This result shows that this multifunctional enzyme unfolds with competition with misfolding in the folding pathway. Comparison of inactivation and conformational changes of the enzyme as well as aggregation imply that unfolding intermediates may exist during urea denaturation. The possible unfolding pathway of fatty acid synthase is also discussed in this paper.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Inactivation kinetics of mushroom tyrosinase by cetylpyridinium chloride

Cetylpyridinium chloride (CPC) was found to inactivate tyrosinase from mushroom (Agaricus bisporus). CPC can bind to the enzyme molecule and induce the enzyme conformation changes. The fluorescence intensity (at 338.4 nm) of the enzyme decreased distinctly with increasing CPC concentrations, and a new little fluorescence emission peak appeared near 372 nm. The inactivation of the enzyme by CPC had first been studied by using the kinetic method of the substrate reaction described by Tsou. The results showed that the enzyme was inactivated by a complex mechanism that had not been previously identified. The enzyme first quickly binds with CPC reversibly and then undergoes a slow irreversible inactivation. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is a saturated trend being independent of CPC concentration if the concentration is sufficiently high. The micro rate constants of inactivation and the association constant were determined.     

A comparative study of ricin and diphtheria toxin-antibody-conjugate kinetics on protein synthesis inactivation

Kinetic data on toxin and antibody-toxin-conjugate inactivation of protein synthesis have been used to assess the variables which affect the transport of these toxins into the cytosol compartment. First-order inactivation rate constants of protein synthesis (ki) are compared under conditions of known receptor occupancy. The effect of inclusion of toxin B chains, both homologous and heterologous, in antibody-toxin conjugates is observed, and factors which affect toxin lag periods are studied. The results show that the inclusion of B chains in conjugates increases ki values 3-10-fold, but only if the B chain is homologous with the A chain. In spite of the augmentation of antibody-toxin-conjugate ki values by homologous toxin B chain, these ki values are only 1/20 those observed with unmodified toxins on sensitive cells. A further difference noted between toxins and antibody-toxin conjugates is the presence of a dose-dependent lag when toxins, but not antibody-toxin conjugates, effect sensitive cell types. This lag period for ricin can be shortened by alkalinizing the cell medium. The kinetic data can be fit by assuming a processing step interposed between the binding of ricin to surface receptors and the interaction of the A chain with ribosomes which is first-order in toxin concentration and pH-dependent. The time constant of this event is reflected in the dose-dependent lag period. It is proposed that antibody-toxin conjugates do not participate in this processing event and therefore fail to achieve the high entry levels exhibited by unmodified toxins.     

Evaluation of epigallocatechin gallate and related plant polyphenols as inhibitors of the FabG and FabI reductases of bacterial type II fatty-acid synthase

Epigallocatechin gallate (EGCG) is the major component of green tea extracts and possesses antibacterial, antiviral, and antitumor activity. Our study focused on validating the inhibition of the bacterial type II fatty acid synthesis system as a mechanism for the antibacterial effects of EGCG and related plant polyphenols. EGCG and the related tea catechins potently inhibited both the FabG and FabI reductase steps in the fatty acid elongation cycle with IC(50) values between 5 and 15 microm. The presence of the galloyl moiety was essential for activity, and EGCG was a competitive inhibitor of FabI and a mixed type inhibitor of FabG demonstrating that EGCG interfered with cofactor binding in both enzymes. EGCG inhibited acetate incorporation into fatty acids in vivo, although it was much less potent than thiolactomycin, a validated fatty acid synthesis inhibitor, and overexpression of FabG, FabI, or both did not confer resistance. A panel of other plant polyphenols was screened for FabG/FabI inhibition and antibacterial activity. Most of these inhibited both reductase steps, possessed antibacterial activity, and inhibited cellular fatty acid synthesis. The ability of the plant secondary metabolites to interfere with the activity of multiple NAD(P)-dependent cellular processes must be taken into account when assessing the specificity of their effects.     

A unique and differential effect of denaturants on cofactor mediated activation of Plasmodium falciparum beta-ketoacyl-ACP reductase

The urea and guanidinium chloride (GdmCl) induced unfolding of FabG, a beta-ketoacyl-ACP reductase of Plasmodium falciparum, was examined in detail using intrinsic fluorescence of FabG, UV-circular dichroism (CD), spectrophotometric enzyme activity measurements, glutaraldehyde cross-linking, and size exclusion chromatography. The equilibrium unfolding of FabG by urea is a multistep process as compared with a two-state process by GdmCl. FabG is fully unfolded at 6.0M urea and 4.0M GdmCl. Approximately 90% of the enzyme activity could be recovered on dialyzing the denaturants, showing that denaturation by both urea and GdmCl is reversible. We found two states in the reversible unfolding process of FabG in presence of NADPH; one is an activity-enhanced state and the other, an inactive state in case of equilibrium unfolding with urea. On the contrary, in presence of NADPH, there is no stabilization of FabG in case of equilibrium unfolding with GdmCl. We hypothesize that the hydrogen-bonding network may be reorganized by the denaturant in the activity-enhanced state formed in presence of 1.0M urea, by interrupting the association between dimer-dimer interface and help in accommodating the larger substrate in the substrate binding tunnel thus, increasing the activity. Furthermore, binding of the active site organizer, NADPH leads to compaction of the FabG in presence of urea, as evident by acrylamide quenching. We have shown here for the first time, the detailed inactivation kinetics of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any of the other sources. These findings provide impetus for exploring the influences of ligands on the structure-activity relationship of Plasmodium beta-ketoacyl-ACP reductase.     

Effect of subunit dissociation, denaturation, aggregation, coagulation, and decomposition on enzyme inactivation kinetics: II. Biphasic and grace period behavior

A model previously developed to characterize enzymatic in activation behavior was used to explain the non-first-order biphasic and grace period phenomena that are often observed with oligomeric enzymes. Luciferase and urease were used as model enzyme such as luciferase, the oligomer initially dissociates reversibly into two native monomer species. These native monomers can then reversibly denature and irreversibly aggregate and coagulate. With the hexamer, urease, two trimers are formed that can subsequently aggregate to form an inactive hexamer. The dissociated monomer species of luciferase do not possess catalytic activity, so the inactivation mechanism, is biphasic; the first slope of a first-order kinetic plot is influenced by the reversible oligomer/monomer/denatured-monomer transition. Whereas the second slope is associated with either irreversible aggregation or coagulation. In contrast, the trimer of urease has the same activity as the hexamer; therefore, during the intitial hexamer-trimer transition, little activity loss occurs. However, as the trimer concentration increases, activity decreases as a result of trimer aggregation. As a result, grace period inactivation behavior is observed. (c) 1992 John Wiley & Sons, Inc.     

Multipoint attachment to a support protects enzyme from inactivation by organic solvents: alpha-Chymotrypsin in aqueous solutions of alcohols and diols

Inactivation of alpha-chymotrypsin in aqueous solutions of alcohols and diols proceeds both reversibly and irreversibly. Reversible loss of the specific enzyme activity results from conformational changes (unfolding) of the enzyme detected by fluorescence spectroscopy. Multipoint covalent attachment to the matrix of polyacryl-amide gel by copolymerization method stabilizes alpha-chymotrypsin from denaturation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. Immobilization protects the enzyme also from irreversible inactivation by organic solvents resulting from bimolecular aggregation and autolysis.     

Kinetics of pressure-induced inactivation of bovine liver glutamate dehydrogenase

Kinetics of pressure-induced denaturation of bovine liver glutamate dehydrogenase (EC 1.4.1.3) were investigated in the pressure range 1.8-2.8 kbar by observing the residual activity after the pressure-release and the scattered light intensity during the incubation at high pressure. The residual activity decreased exponentially with the incubation time, whereas the scattered light intensity showed a bimodal profile indicating parallel aggregation and dissociation reactions. The latter suggested that two kinds of aggregates were formed during the incubation under pressure. The observed first-order rate constant for the inactivation, k obs, showed a minimum around 30 degrees C. These experimental results were interpreted in terms of the following reaction scheme; (formula; see text) where N represents the enzyme entity with native structure, D1 the partially denatured intermediate, D2 the irreversibly denatured state, and A1 and A2 the two kinds of aggregates, one of which (A1) is reversibly formed at an early stage of the incubation under high pressure. The apparent activation volume for the inactivation reaction was estimated to be delta V*app = -113 +/- 5 cm3 X mol-1 from the pressure dependence of k obs. The effect of coenzyme, NAD+, on the pressure-induced inactivation was also studied. The inactivation was retarded by the presence of the coenzyme, whereas the apparent activation volume for the holoenzyme (delta V*app = -104 +/- 2 cm3 X mol-1) did not differ significantly from that for the apoenzyme.     

Stopped-flow studies of myelin basic protein association with phospholipid vesicles and subsequent vesicle aggregation

When mixed with vesicles containing acidic phospholipids, myelin basic protein causes vesicle aggregation. The kinetics of this vesicle cross-linking by myelin basic protein was investigated by using stopped-flow light scattering. The process was highly cooperative, requiring about 20 protein molecules per vesicle to produce a measurable aggregation rate and about 35 protein molecules per vesicle to produce the maximum rate. The maximum aggregation rate constant approached the theoretical vesicle-vesicle collisional rate constant. Vesicle aggregation was second order in vesicle concentration and was much slower than protein-vesicle interaction. The highest myelin basic protein concentration used here did not inhibit vesicle aggregation, indicating that vesicle cross-linking occurred through protein-protein interactions. In contrast, poly(L-lysine)-induced vesicle aggregation was easily inhibited by increasing peptide concentrations, indicating that it did cross-link vesicles as a peptide monomer. The myelin basic protein:vesicle stoichiometry required for aggregation and the low affinity for protein dimerization suggested that multiple protein cross-links were needed to form a stable aggregate. Stopped-flow fluorescence was used to estimate the kinetics of myelin basic protein-vesicle binding. The half-times obtained suggested a rate constant that approached the theoretical protein-vesicle collisional rate constant.     

Aggregation of an amyloidogenic fragment of human islet amyloid polypeptide

Native human islet amyloid polypeptide (hIAPP) has been identified as the major component of amyloid plaques found in the pancreatic islets of Langerhans of persons affected by type 2 diabetes mellitus. Early studies of hIAPP determined that a segment of the molecule, amino acids 20-29, is responsible for its aggregation into amyloid fibrils. The present study demonstrates that the aggregation of hIAPP 20-29-Trp is a nucleation-dependent process, displaying a distinct lag time before the onset of rapid aggregation. Moreover, the lag time can be eliminated by seeding the sample of unaggregated peptide with preformed fibrils. In contrast to the expectation from the conventional model of nucleation-dependent aggregation, however, the lag time of hIAPP aggregation does not depend on peptide concentration. To explain this observation, a modified version of the standard model of nucleation-dependent aggregation is presented in which the monomeric peptide concentration is buffered by an off-aggregation-pathway formation of peptide micelles.     

Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase by zinc ions: a new type of complexing inhibition

The Tsou method was used to study the kinetic course of inactivation of green crab alkaline phosphatase by zinc ions. The results show that the enzyme was inactivated by a complexing scheme which has not been previously identified. The enzyme first reversibly and quickly binds Zn(2+) and then undergoes a slow reversible course to inactivation and slow conformational change. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is for a saturated reaction being independent of Zn(2+) concentration if the concentration is sufficiently high. The microscopic rate constants of inactivation and the association constant were determined from the measurements.     

Hysteretic enzyme response induced by inhibitory antibodies against human leukocyte elastase

Polyclonal antibodies raised in rabbits against human leukocyte elastase contained two distinct populations of enzyme-inhibiting immunoglobulins. The enzyme-catalyzed reaction in the presence of antibodies (both IgG or monovalent Fab fragments) showed a transient state lasting up to several minutes depending on the inhibitor and substrate concentrations, which was followed by a linear steady-state. The transient was a concave upward or concave downward lag phase depending on whether the enzyme had been preincubated with the antibodies or not, respectively. The kinetic analysis of reaction progress curves showed that both antibody populations were slow inhibitors, which completely and reversibly excluded the substrate from binding to the enzyme. For both antibody populations, the formation of the enzyme-inhibitor complex was characterized by an initial rapid interaction followed by a slow isomerization to a catalytically inactive complex. The apparent pseudo first-order rate constant of the transient slow phase was a hyperbolic function of the inhibitor concentration for both antibodies, from which relevant kinetic constants and the half times for enzyme inactivation could be calculated. For instance, with a total antibody concentration of 1 mg/ml (as IgG), leukocyte elastase was inactivated with t1/2 = 0.31s and 24.8s by the faster and the slower of the two antibodies, respectively. It is suggested that the hysteretic response of the enzyme to the inhibitory action of its antibodies may be due to a kind of memory of the antibody molecule for a special inactive enzyme conformation resulting from inhibition by proteinase inhibitors during the immunization procedure. In turn, the purified antibodies would be able to reversibly induce a slow transition of the enzyme molecule from an active to a substrate-excluding conformation ("induced misfit").     

In vitro aggregation of platelets induced by alpha-toxin (phospholipase C) of Clostridium perfringens.

Highly purified alpha-toxin (phospholipase C) of Clostridium perfringens prepared by affinity chromatography on agarose-linked egg-yolk lipoprotein induced the in vitro aggregation of platelets of an irreversible type. The aggregation started after a time lag, the length of which depended on the concentration of the toxin; the reciprocal of the time lag was found to be directly proportional to the toxin concentration. Using this assay method, we demonstrated that the platelet-aggregating activity of alpha-toxin reached minimum at around 70 C but heating at higher temperatures inactivated it to a lesser extent; the same anomaly in heat inactivation was observed with phospholipase C activity possessed by the toxin. By subjecting purified alpha-toxin to isoelectric focusing, four molecular forms were isolated, all of which were associated with both the platelet-aggregating and phospholipase C activities. From all these results we concluded that the entity responsible for the platelet-aggregating activity is identical with alpha-toxin (phospholipase C).     

Cyanide inactivation of hydrogenase from Azotobacter vinelandii.

The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M-1 min-1 for CN-). The rate of inactivation decreased with decreasing pH. [14C]cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.     

Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.