Biomedical subjects

R Varón

Publications and source records attributed to R Varón.

At least 37 records · Page 2Linked to original sources

PubMed2000

Use of a windows program for simulation of the progress curves of reactants and intermediates involved in enzyme-catalyzed reactions.

A program that performs simulation of the kinetics of enzyme-catalyzed reactions with up to 32 species is described. The program is written in C++ for MS Windows 95/98/NT and uses a simple text file to define the kinetic model. The use of the program is illustrated with some examples. WES is available free of charge on request from the authors (e-mail: fgarcia@iele-ab.uclm.es).

Catalysis↗

PubMed2000

Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase.

This paper reports a quantitative study of the effect of ring substituents in the 1-position of the aromatic ring on the rate of monophenol hydroxylation and o-diphenol oxidation catalyzed by tyrosinase. A possible correlation between the electron density of the carbon atom supporting the oxygen from the monophenolic hydroxyl group and the V Mmax values for each monophenol was found. In the case of o-diphenols the same effect was observed but the size of the side-chain became very important. NMR studies on the monophenols justified the sequence of the V Mmax values obtained. As regards the o-diphenols, on the other hand, only a fair correlation between NMR and V Dmax values was observed due to the effect of the molecular size of the ring substituent. From these data, it can be concluded that the redox step (k33) is not the rate-determining step of the reaction mechanism. Thus, the monophenols are converted into diphenols, but the order of specificities towards monophenols is different to that of o-diphenols. The rate-limiting step of the monophenolase activity could be the nucleophilic attack (k51) of the oxygen atom of the hydroxyl group on the copper atoms of the active site of the enzyme. This step could also be similar to or have a lower rate of attack than the electrophilic attack (k52) of the oxygen atom of the active site of oxytyrosinase on the C-3 of the monophenolic ring. However, the rate-limiting step in the diphenolase activity of tyrosinase could be related to both the nucleophilic power of the oxygen atom belonging to the hydroxyl group at the carbon atom in the 3-position (k32) and to the size of the substituent side-chain. On the basis of the results obtained, kinetic and structural models describing the monophenolase and diphenolase reaction mechanisms for tyrosinase are proposed.

Agaricales↗

PubMed2000

Oxidation by mushroom tyrosinase of monophenols generating slightly unstable o-quinones.

Tyrosinase can act on monophenols because of the mixture of mettyrosinase (Em) and oxytyrosinase (Eox) that exists in the native form of the enzyme. The latter form is active on monophenols although the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex as the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as 4-methoxyphenol, 4-ethoxyphenol and 4-tert-butylphenol, tyrosinase generates o-quinones which become unstable with small constants of approximately < 10-3 s-1. The system evolves from an initial steady state, reached when t-->0, through a transition state towards a final steady state, which is never reached because the substrate is largely consumed. The mechanisms proposed to explain the enzyme's action can be differentiated by the kinetics of the first steady state. The results suggest that tyrosinase hydroxylates monophenols to o-diphenols, generating an intermediate Em-diphenol in the process, which may oxidize the o-diphenol or release it directly into the medium. In the case of o-quinone formation, its slow instability generates o-diphenol which activates the enzymatic system yielding parabolic time recordings.

Agaricales↗

PubMed2000

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols.

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding constant decreases, leading to an increase in K(M)m. The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V(M)max. The small difference between the absolute values of V(M)max suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.

Agaricales↗

PubMed1999

Transient phase of enzyme reactions. Time course equations of the strict and the rapid equilibrium conditions and their computerized derivation.

In this contribution, we present the derivation, from the strict transient phase equations of enzyme reactions, of the transient phase equations under the usual assumptions that one or more of the reversible steps involved in the mechanism of the enzyme reaction are assumed to be in rapid equilibrium. Moreover, we present the transient phase equations of all of the species in a general enzyme system model, valid for the partial or total rapid equilibrium conditions, as well as the particular case of the strict transient phase equations. In the case of the rapid equilibrium assumptions, the equations may be given either as functions of the individual rate constants in the reversible steps assumed in rapid equilibrium or as functions of the corresponding equilibrium constants. The steady state equations are easily obtained from the transient phase equations by setting the time --> infinity. We have implemented a computer program, easy to use and with a user-friendly format for the input of data and output of results, which allows the user to derive the symbolic strict transient phase equations and/or those corresponding to the assumption that one or more of the reversible reaction steps are in rapid equilibrium.

Computer Simulation↗

PubMed1999

Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.

The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.

Catalysis↗

PubMed1998

Kinetics of enzyme systems with unstable suicide substrates.

This paper deals with kinetic studies of enzyme reaction mechanisms with enzyme inactivation induced by an unstable suicide substrate. An initial steady-state of the catalytic route is assumed and the time course equations for the total active enzyme forms and the reaction product have been derived. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters is suggested and the time course equations of an important reaction mechanisms involving a stable suicide substrate and which can be regarded as particular case of that under study has also been derived from the corresponding equations. The simplicity of our method allows its systematic application to more complex mechanisms.

Enzyme Inhibitors↗

PubMed1997

Kinetic study of the oxidation of 4-hydroxyanisole catalyzed by tyrosinase.

Despite the importance of the substrate 4-hydroxyanisole in melanoma therapy, the kinetics of its oxidation catalyzed by tyrosinase has never been properly characterized. This approach is reported here for the first time. The applicability to 4-hydroxyanisole of the reaction mechanism of tyrosinase previously proposed for other monophenols has been corroborated. The Michaelis constant for the oxidation of 4-hydroxyanisole catalyzed by mushroom tyrosinase was (62 +/- 1.5) microM at pH 7 and increased when the pH decreased, reaching a value of (195 +/- 5) microM at pH 5.5. However the maximum steady-state rate, whose value was (0.54 +/- 0.01) microM/min, did not change with the pH. The apparent catalytic constant was (184 +/- 5) s-1, around twenty three times higher than that previously described for L-tyrosine (8 s-1).

Anisoles↗

PubMed1996

Kinetics of an autocatalytic zymogen reaction in the presence of an inhibitor coupled to a monitoring reaction.

A global kinetic analysis of a model consisting of an autocatalytic zymogen-activation process, in which an irreversible inhibitor competes with the zymogen for the active site of the proteinase, and a monitoring coupled reaction, in which the enzyme acts upon one of its substrates, is presented. This analysis is based on the progress curves of any of the two products released in the monitoring reaction. The general solution is applied to an important particular case in which rapid equilibrium conditions prevail. Finally, we suggest a procedure to predict whether the inhibition or activation route dominates in the steady state of the system. These results generalize our previous analysis of simpler mechanisms.

Binding Sites↗

PubMed1996

Oxymetric and spectrophotometric study of the ascorbate oxidase activity shown by frog epidermis tyrosinase.

Many studies concerning the effect of ascorbic acid on the action of tyrosinase on several substrates have been carried out with contradictory results. The results shown in this work comprise a hypothetical reaction mechanism, which explains the ascorbate oxidase activity of frog epidermis tyrosinase. The reaction between frog epidermis tyrosinase and L-ascorbic acid was studied by oxymetric and spectrophotometric assays. The activity was linearly related to enzyme concentration, with a Michaelis constant for L-ascorbic acid of 0.160 +/- 0.009 mM and Vmax of 90 +/- 4 nM/s. Maximum activity was obtained at pH 7.5. The stoichiometry of the reaction was calculated by measuring the substrate (O2 and L-ascorbic acid) consumption as well as the initial rates of the consumption of oxygen and the disappearance of L-ascorbic acid. The stoichiometry was found to be 1:2 (O2:L-ascorbic acid). The action of the tyrosinase inhibitor tropolone was also studied. All the results present evidence concerning the ascorbate oxidase activity of frog epidermis tyrosinase and a possible reaction mechanism based on the different enzymatic forms of tyrosinase to explain such activity.

Animals↗

PubMed1995

A continuous spectrophotometric method for determining the monophenolase and diphenolase activities of apple polyphenol oxidase.

A continuous spectrophotometric method for the determination of the monophenolase and diphenolase activities of apple polyphenol oxidase is described. The method is based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone (MBTH) and the quinone product of the oxidation of p-hydroxyphenyl propionic acid and 3,4-dihydroxyphenyl propionic acid in the presence of polyphenol oxidase. The lambda(max) and molar absorptivity (epsilon) for the MBTH-quinone adduct have been calculated. The presence of MBTH in the reaction medium decreases the lag period during the expression of monophenolase activity. The high value of V(mas) suggests the existence of a high catalytic constant. This, together with the value of epsilon for the MBTH-quinone adduct, makes this method more sensitive than other continuous methods.

Catechol Oxidase↗

PubMed1995

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response.

Alkaline Phosphatase↗

PubMed1995

General linear compartment model with zero input: I. Kinetic equations.

The derivation of kinetic equations is described for n-compartment linear models, in which the substance may be simultaneously introduced into one or more compartments at t = 0 and eliminated from any compartment. For a given zero-input, general formulas are derived which describe the amount of tracer in any of the compartments as a function of time and the model parameters. New algorithms have been developed which allow the expression of the kinetic equations.

Algorithms↗

PubMed1995

General linear compartment model with zero input: III. First passage residence time of enzyme systems.

In this paper, we present an alternative procedure to derive the residence times of enzyme and compartment systems. This procedure allows to express the residence time by a general, symbolic and simplified formula relating it directly with the rate constants. It is applicable to any enzyme reaction scheme which can be formulated as a set of first-order or pseudo-first order interconversions, without any other restriction. A computer program has been developed that greatly facilitates the task of the residence time derivations. The above analysis was extrapolated to any linear compartment model.

Computer Simulation↗

PubMed1995

General linear compartment model with zero input: II. The computerized derivation of the kinetic equations.

The final equations obtained in the first article of this series describing the concentrations of substances in any of the compartments of a model are here cast in an easily programmable form. A computer program with an easy input method and the ability to expand all of the coefficients in the kinetic equations in terms of the model parameters has been developed. The program has been written in the BASIC language.

Computer Simulation↗

PubMed1995

Kinetic analysis of an autocatalytic process coupled to a reversible inhibition: the inhibition of the system trypsinogen-trypsin by p-aminobenzamidine.

A kinetic analysis of the mechanism of autocatalytic activation in the presence of a reversible inhibitor is presented. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. We have extended the kinetic equations derived to a particular case in rapid equilibrium conditions. This analysis is illustrated by the experimental study of the inhibition by p-aminobenzamidine of trypsin activity in its action on trypsinogen. In such system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The results obtained show that the apparent activation rate constant decreases non-linearly with the initial concentration of inhibitor, according to the equations obtained in the kinetic analysis.

Benzamidines↗

PubMed1994

Kinetics of an enzyme reaction in which both the enzyme-substrate complex and the product are unstable or only the product is unstable.

A kinetic analysis of the Michaelis-Menten mechanism has been made for the case in which both the enzyme-substrate complex and the product are unstable or only the product is unstable, either spontaneously or as the result of the addition of a reagent. This analysis allows the derivation of equations which under conditions of limiting enzyme concentration relate the concentration of all of the species to the time. A kinetic data analysis is suggested, which leads to the evaluation of the kinetic parameters involved in the reaction. The analysis is based on the equation which describes the formation of products with time and one's experimental progress curves. We demonstrate the method numerically by computer simulation of the reaction with added experimental errors and experimentally by the use of data from the kinetic study of the action of tyrosinase on dopamine.

Basidiomycota↗

PubMed1994

Kinetics study of the oxidation of 4-tert-butylphenol by tyrosinase.

The reaction between 4-tert-butylphenol (BuPhOH) and mushroom tyrosinase was investigated by following 4-tert-butyl-ortho-benzoquinone, whose high stability permits the reaction to be used as a model for the study of the monophenolase activity of tyrosinase. The system evolves to a pseudo-steady state through an induction period (tau), the pseudo-steady-state rate (Vss) decreasing when the (BuPhOH) concentration increases. Increases in enzyme concentration result in a parabolic pattern with Vss, while tau is shortened. The addition of increasing catalytic amounts of 4-tert-butylcatechol at the start of the reaction reduces tau until it is totally abolished, an initial burst being observed at high 4-t-butylatechol concentrations. Initial bursts are also obtained at pH 4.5 or lower, indicating a lower affinity of the met-tyrosinase or oxidized form for the monophenol at low pH. These experimental results can be explained by the reaction mechanism of tyrosinase.

Basidiomycota↗