A kinetic study of the suicide inactivation of an enzyme measured through coupling reactions. Application to the suicide inactivation of tyrosinase.

A systematic procedure for the kinetic study of reaction mechanisms with enzyme inactivation induced by a suicide substrate in the presence or in the absence of an auxiliary substrate, when the enzyme activity is measured through coupling reactions, enzymically catalysed or not, was developed and analysed by using the transient-phase approach. The methodology is established to determine the parameters and kinetic constants corresponding to the enzyme suicide inactivation and the coupling reactions. This approach is illustrated by a study of the suicide inactivation of tyrosinase by catechol in the presence of L-proline. Treatment of the experimental data was carried out by non-linear regression.     

Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants

The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.     

Inhibition kinetics of mushroom tyrosinase by copper-chelating ammonium tetrathiomolybdate

With a strategy of chelating coppers at tyrosinase active site to detect an effective inhibitor, several copper-specific chelators were applied in this study. Ammonium tetrathiomolybdate (ATTM) among them, known as a drug for treating Wilson's disease, turned out to be a significant tyrosinase inhibitor. Treatment with ATTM on mushroom tyrosinase completely inactivated enzyme activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway and dilution of the ATTM revealed that ATTM is a tight-binding inhibitor and high dose of ATTM irreversibly inactivated tyrosinase. Progress-of-substrate reaction kinetics and activity restoration with a dilution of the ATTM indicated that the copper-chelating ATTM may bind slowly but reversibly to the active site without competition with substrate, and the enzyme-ATTM complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. Thus, inhibition by ATTM on tyrosinase could be categorized as complexing type of inhibition with a slow and reversible binding. Detailed analysis of inhibition kinetics provided IC50 at the steady-state and inhibitor binding constant (K(I)) for ATTM as 1.0+/-0.2 microM and 10.65 microM, respectively. Our results may provide useful information regarding effective inhibitor of tyrosinase as whitening agents in the cosmetic industry.     

Inhibitory effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase

Mushroom tyrosinase (EC 1.14.18.1) is a copper containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay was performed in air-saturated solutions and the kinetic behavior of this enzyme in the oxidation of L-tyrosine and L-DOPA has been studied. The effects of cupferron on the monophenolase and diphenolase activity of mushroom tyrosinase have been studied. The results show that cupferron can inhibit both monophenolase and diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activity of the enzyme decreased sharply. Cupferron can lead to reversible inhibition of the enzyme, possibly by chelating copper at the active site of the enzyme. The IC(50) value was estimated as 0.52 microM for monophenolase and 0.84 microM for diphenolase. A kinetic analysis shows that the cupferron is a competitive inhibitor for both monophenolase and diphenolase. The apparent inhibition constant for cupferron binding with free enzyme has been determined to be 0.20 microM for monophenolase and 0.48 microM for diphenolase.     

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.     

Kinetic study of a substrate cycle involving a chemical step: highly amplified determination of phenolic compounds

A kinetic analysis of a substrate cycle in which one of the two steps was substituted by a chemical reaction has been made. The model is illustrated by the amplified determination, in a continuous assay, of phenolic compounds at low concentrations using the enzyme tyrosinase and β-NADH to reduce the o-quinone product of catalytic activity. Progress curves corresponding to β-NADH disappearance were not linear and followed first-order kinetics. Knowledge of the kinetics of the reaction has allowed us to achieve detection limits as low as 50 nM in a simple 10-min assay. There is no analytical solution to the non-linear differential equation system that describes the kinetics of the reaction, therefore, computer simulations of its dynamic behaviour are also presented, good agreement with the experimental results being obtained. The method is applicable to the measurement of any other metabolite, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be implemented in a bioreactor for automation purposes.     

Irreversibly inhibitory kinetics of 3,5-dihydroxyphenyl decanoate on mushroom (Agaricus bisporus) tyrosinase

3,5-Dihydroxyphenyl decanoate (DPD) is found to inhibit the diphenolase activity of tyrosinase from mushroom (Agaricus bisporus). The effects of DPD on the diphenolase activity of mushroom tyrosinase have been studied. The results show that the enzyme activity decreases very slowly with an increase in DPD concentrations at lower concentrations of DPD (between 5 and 60 microM). But at higher concentrations of DPD, DPD can strongly inhibit the diphenolase activity of the enzyme and the inhibition is irreversible. The IC50 value was estimated to be 96.5 microM. The inhibition mechanism of DPD has been investigated and the results show that DPD can bind to the free enzyme molecule and enzyme-substrate complex and lose the enzyme activity completely. The inhibition kinetics has been studied in detail by using the kinetic method of the substrate reaction described by Tsou. The microscopic rate constants of the enzyme inhibited by DPD at higher concentrations have been determined.     

Kinetic characterization of dopamine as a suicide substrate of tyrosinase

A kinetic study of the inactivation of frog epidermis tyrosinase by a suicide substrate dopamine hydrochloride is described. The kinetic parameters and constants which characterize this reaction have been determined and the effects of pH and the stoichiometric inhibition by chloride have been considered.     

Tyrosinase inactivation in organic solvents.

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction.     

pH-dependent interconvertible forms of mushroom tyrosinase with different kinetic properties

The lag in cresolase activity and inhibition by excess tyrosine of mushroom tyrosinase which was observed when assayed at pH 6.8 was found to be absent when assayed at pH 5.0. The absence of lag and inhibition by excess tyrosine of tyrosinase at pH 5.0 were brought about only after the enzyme was kept at pH 5.0, at 0-4 degrees C, for 1.5 h. The enzyme kept at pH 5.0 for 1.5-3 h at 0-4 degrees C when brought back to pH 6.8, acquires lag and inhibition by excess tyrosine when its activity was measured at pH 6.8. The pH-dependent changes in the kinetic properties of the mushroom tyrosinase are similar to the pH-dependent changes in the kinetic properties of tyrosinase from B-16 murine melanoma and human skin, and thus appear to be a general property of tyrosinase from diverse sources.     

Inactivation kinetics of mushroom tyrosinase in the dimethyl sulfoxide solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of copper-containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones and then forms brown or black pigments. In the present paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC ₅₀ is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.     

Kinetics of thermal inactivation of penaeus penicillatus acid phosphatase

The kinetics of thermal inactivation of Penaeus penicillatus acid phosphatase have been studied using a kinetic method related to the substrate reaction during irreversible inhibition of the enzyme activity as previously described by Tsou (Adv. Enzymol. Relat. Areas Mol. Biol. (1988) 61, 381-436). The kinetics of thermal inactivation of the enzyme show that the reaction is irreversible. The microscopic rate constants were determined for thermal inactivation of free enzyme and the enzyme--substrate complex. The results show that the presence of substrate has a significant protective effect against thermal inactivation of the enzyme.     

Pyruvate Kinase Activity in Crude Extracts of Leaves of Cynodon dactylonand Other C4Plants

A new extraction procedure and an LDH-coupled assay method are presented for the study of pyruvate kinase (PK) in leaf crude extracts from Cynodon dactylon(L.) Pers and other C₄plants. Extraction at pH 6.8 and assay at pH 6.2 facilitated the measuring of PK activity by eliminating phosphoenolpyruvate carboxylase interference more effectively than the thermal inactivation or chemical inhibition previously used. The method suggested did not affect the kinetic properties of PK as compared to the purified enzyme from C. dactylon.     

Inhibitory kinetics of quercetin on phenoloxidase from loopworm

Abstract Phenoloxidase (PO; monophenol or dihydroxyphenolalanine: oxygen oxidoreductase; EC. 1.14.18.1 and also known as tyrosinase) is one of the key enzymes in the insect moulting process, so screening based on discovery of inhibitors of the enzyme might ultimately provide clues for enhancing the control of insect pests. In the present investigation, the inactivation of phenoloxidase from loopworm Semiothisa cinerearia Bremer et Grea (Homoptera: Geometridae) by quercetin were studied. The results showed that low concentration to 180 αmol/L quercetin could inhibit PO activity, and the IC₅₀ (inhibition concentration showing 50% of the maximum inhibition) was estimated to be 111.5 αmol/L. In addition, quercetin was proven to be a competitive inhibitor, and the equilibrium constant for the inhibitor binding was determined. The results also showed that inactivation of the enzyme by quercetin was a slow, reversible reaction with fractional remaining activity, and the microscopic reaction rate constants of the inhibitor with the enzyme were determined.     

Modification of lipoxygenase by hydrogen peroxide and photooxidation.

The kinetic study of fluorescence stopped-flow method suggested that the interaction between lipoxygenase and H2O2 is consistent with a simple irreversible one-step mechanism. The activation energy of the reaction was 7.2 kcal/mol. Participation of an ionizable group with pK about 8.8, possibly a histidine residue, was suggested from the pH-dependence of the rate constant. No further fluorescence quenching of lipoxygenase was observed when the product was added to the lipoxygenase solution before mixing the lipoxygenase and H2O2 solutions. The fluorescence quenching of lipoxygenase by H2O2 was in parallel with the inactivation of the enzyme. Hydroperoxylinoleic acid strongly protects the inactivation of lipoxygenase caused by H2O2. These results are consistent with an interpretation that OH- and/or O- - are produced when the iron of the enzyme is oxidized by H2O2, which in turn will attack some amino acid essential for the enzyme activity. The pH-dependence of the inactivation rate constant of photooxidation of lipoxygenase sensitized by methylene blue indicated that an ionizable group with pK about 8.8 is concerned with the enzymatic activity. In contrast to the inactivation of lipoxygenase by H2O2, the product protected the inactivation of the enzyme by photooxidation only at high concentration.     

Kinetics of inactivation of phytase (phy A) during modification of histidine residue by IAA and DEP

Chemical probing of histidine residues using specific modifiers, iodoacetic acid (IAA) and diethylpyrocarbonate (DEP) resulted in the inactivation of phytase (phy A). The kinetic theory of the substrate reaction during the modification of enzyme activity was applied to a study of the kinetics of the course of inactivation of phytase by IAA and DEP. The results suggested that histidine residues are involved in the active site of the enzyme. They also indicated that inactivation of the enzyme by IAA was via a complexing type inhibition, while the inhibition by DEP reaction involved a conformational change step before inactivation. The dissociation constant of the enzyme-inhibitor complex of IAA, the constant of the conformational change of DEP and the microscopic rate constants of two inhibitors were obtained.     

Kinetics of inactivation of Ulva pertusa Kjellm alkaline phosphatase by ethylenediaminetetraacetic acid disodium

Ulva pertusa Kjellm alkaline phosphatase (EC 3.3.3.1) is a metalloenzyme, the active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory described by Tsou of the substrate reaction during irreversible inhibition of enzyme activity has been employed to study the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction at different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA indicated a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing that the initial formation of an enzyme-EDTA complex is a relative rapid reaction, following by a slow inactivation step that probably involves a conformational change of the enzyme. The presence of Zn2+ apparently stabilizes an active-site conformation required for enzyme activity.     

Half-time analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific reagent

The half-time method for the determination of Michaelis parameters from enzyme progress-curve data (Wharton, C.W. and Szawelski, R.J. (1982) Biochem. J. 203, 351-360) has been adapted for analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific inhibitor. The method is applicable to a model in which a product (R) of the decomposition of the site-specific reagent, retaining the chemical moiety responsible for inhibitor specificity, binds reversibly to the enzyme with dissociation constant Kr: (formula; see text). Half-time plots of simulated enzyme inactivation time-course data are shown to be unbiased, and excellent estimates of the apparent second-order rate constant for inactivation (k +2/Ki) and Kr can be obtained from a series of experiments with varying initial concentrations of inhibitor. Reliable estimates of k +2 and Ki individually are dependent upon the relative magnitudes of the kinetic parameters describing inactivation. The special case, Kr = Ki, is considered in some detail, and the integrated rate equation describing enzyme inactivation shown to be analogous to that for a simple bimolecular reaction between enzyme and an unstable irreversible inhibitor without the formation of a reversible enzyme-inhibitor complex. The half-time method can be directly extended to the kinetics of enzyme inactivation by an unstable mechanism-based (suicide) inhibitor, provided that the inhibitor is not also a substrate for the enzyme.     

Tyrosinase inhibition due to interaction of homocyst(e)ine with copper: the mechanism for reversible hypopigmentation in homocystinuria due to cystathionine beta-synthase deficiency.

Deficiency of cystathionine beta-synthase (CBS) is a genetic disorder of transsulfuration resulting in elevated plasma homocyst(e)ine and methionine and decreased cysteine. Affected patients have multisystem involvement, which may include light skin and hair. Reversible hypopigmentation in treated homocystinuric patients has been infrequently reported, and the mechanism is undefined. Two CBS-deficient homocystinuric patients manifested darkening of their hypopigmented hair following treatment that decreased plasma homocyst(e)ine. We hypothesized that homocyst(e)ine inhibits tyrosinase, the major pigment enzyme. The activity of tyrosinase extracted from pigmented human melanoma cells (MNT-1) that were grown in the presence of homocysteine was reduced in comparison to that extracted from cells grown without homocysteine. Copper sulfate restored homocyst(e)ine-inhibited tyrosinase activity when added to the culture cell media at a proportion of 1.25 mol of copper sulfate per 1 mol of DL-homocysteine. Holo-tyrosinase activity was inhibited by adding DL-homocysteine to the assay reaction mixture, and the addition of copper sulfate to the reaction mixture prevented this inhibition. Other tested compounds, L-cystine and betaine did not affect tyrosinase activity. Our data suggest that reversible hypopigmentation in homocystinuria is the result of tyrosinase inhibition by homocyst(e)ine and that the probable mechanism of this inhibition is the interaction of homocyst(e)ine with copper at the active site of tyrosinase.     

Kinetics of inactivation of green crab (Scylla Serrata) alkaline phosphatase during removal of zinc ions by ethylenediaminetetraacetic acid disodium

Green crab (Scylla Serrata) alkaline phosphatase (EC 3.1.3.1.) is a metalloenzyme, the each active site in which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied to a study on the kinetics of the course of inactivation of the enzyme by ethylenediaminetetraacetic acid disodium (EDTA). The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenyl phosphate (PNPP) and inactivator EDTA suggested a complexing mechanism for inactivation by, and substrate competition with, EDTA at the active site. The inactivation kinetics are single phasic, showing the initial formation of an enzyme-EDTA complex is a relatively rapid reaction, followed a slow inactivation step that probably involves a conformational change of the enzyme. Zinc ions are finally removed from the enzyme. The presence of metal ions apparently stabilizes an active-site conformation required for enzyme activity.