Biomedical subjects

F García-Cánovas

Publications and source records attributed to F García-Cánovas.

At least 19 recordsLinked to original sources

PubMed2006

Expressions for the fractional modification in different monocyclic enzyme cascade systems: analysis of their validity tested by numerical integration.

This paper presents the derivation, under a minimal set of assumptions, of a general expression for the steady-state fractional modification of an interconvertible protein involved in four different schemes of monocyclic enzyme cascade systems. From this general expression we derive, as particular cases, other, simpler expressions by applying additional assumptions and which have, therefore, a smaller range of validity. Some of these particular expressions coincide with those already obtained in previous contributions on individualised analyses. We discuss the relationships between the kinetic parameters and the concentrations needed for the fulfilment of the additional assumptions. The goodness of the analysis was tested by reference to the shape in the steady-state of the simulated time progress curves obtained by numerical integration.

Algorithms↗

PubMed2006

Kinetic analysis of a general model of activation of aspartic proteinase zymogens.

Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.

Animals↗

PubMed2006

Calculating molar absorptivities for quinones: application to the measurement of tyrosinase activity.

The molar absorptivities of the quinones produced from different o-diphenols, triphenols, and flavonoids were calculated by generating the respective quinones through oxidation with an excess of periodate. Oxidation of these substrates by this reagent was analogous to oxidation by tyrosinase with molecular oxygen, although the procedure showed several advantages over the enzymatic method in that oxidation took place almost immediately and quinone stability was favored because no substrate remained. The o-diphenols studied were pyrocatechol, 4-methylcatechol, 4-tert-butylcatechol, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethylamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, and caffeic acid; the triphenols studied were pyrogallol, 1,2,4-benzenetriol, 6-hydroxydopa, and 6-hydroxydopamine; and the flavonoids studied were (+)catechin, (-)epicatechin, and quercetin. In addition, the stability of the quinones generated by oxidation of the compounds by [periodate]0/[substrate]0 << 1 was studied. Taking the findings into account, tyrosinase could be measured by following o-quinone formation in rapid kinetic studies using the stopped-flow method. However, measuring o-quinone formation could not be useful for steady-state studies. Therefore, several methods for following tyrosinase activity are proposed, and a kinetic characterization of the enzyme's action on these substrates is made.

Benzoquinones↗

PubMed2005

Enzymatic method with polyphenol oxidase for the determination of cysteine and N-acetylcysteine.

Thiols, such as cysteine and N-acetylcysteine, are included in many pharmaceutical products for their mucolytic properties. The method described here uses mushroom polyphenol oxidase (PPO) to determine two thiols and consists of measuring the lag period in the formation of the product generated as PPO acts on o-diphenol in the presence of a thiol. In the experimental conditions, o-quinone is formed enzymatically and then reacts stoichiometrically with the thiol, originating the corresponding thiol-diphenol adduct, which does not absorb visible light. Once the thiol has been used up, the o-quinone can be observed in the medium. It must be borne in mind that the inhibition of PPO is practically null at low concentrations of thiol, and the only effect observed is the formation of the thiol-diphenol adduct. In the following, an exact kinetic method capable of rapidly and accurately assaying thiols with PPO and o-diphenol is optimized and is shown to be a straightforward way of calculating thiol concentration. The method has been successfully applied to the determination of cysteine in model solutions and of N-acetylcysteine in pharmaceutical products.

Acetylcysteine↗

PubMed2005

The influence of product instability on slow-binding inhibition.

We present a kinetic study of an enzyme reaction that takes place with slow-binding inhibition where the immediate product undergoes a spontaneous or induced process of decomposition. A kinetic study of an enzyme process, in which a slow-binding inhibition process and a decomposition of the immediate product of the reaction take place simultaneously is performed. The corresponding explicit concentration-time equations were obtained. Using the analytical solutions obtained, which were tested numerically, we suggest a procedure that allows the discrimination between the particular cases considered and the evaluation of the principal kinetic parameters of the reaction.

Enzyme Inhibitors↗

PubMed2004

Tyrosinase kinetics: discrimination between two models to explain the oxidation mechanism of monophenol and diphenol substrates.

The kinetic behaviour of tyrosinase is very complex because the enzymatic oxidation of monophenol and o-diphenol to o-quinones occurs simultaneously with the coupled non-enzymatic reactions of the latter. Both reaction types are included in the kinetic mechanism proposed for tyrosinase (Mechanism I [J. Biol. Chem. 267 (1992) 3801-3810]). We previously confirmed the validity of the rate equations by the oxidation of numerous monophenols and o-diphenols catalysed by tyrosinase from different fruits and vegetables. Other authors have proposed a simplified reaction mechanism for tyrosinase (Mechanism II [Theor. Biol. 203 (2000) 1-12]), although without deducing the rate equations. In this paper, we report new experimental work that provides the lag period value, the steady-state rate, o-diphenol concentration released to the reaction medium. The contrast between these experimental data and the respective numerical simulations of both mechanisms demonstrates the feasibility of Mechanism I. The need for the steps omitted from Mechanism II to interpret the experimental data for tyrosinase, based on the rate equations previously deduced for Mechanism I is explained.

Agaricales↗

PubMed2003

Mean residence times in linear compartmental systems. Symbolic formulae for their direct evaluation.

A complete analysis has been performed of the mean residence times in linear compartmental systems, closed or open, with or without traps and with zero input. This analysis allows the derivation of explicit and simple general symbolic formulae to obtain the mean residence time in any compartment of any linear compartmental system, closed or open, with or without traps, as well as formulae to evaluate the mean residence time in the entire system like the above situations. The formulae are given as functions of the fractional transfer coefficients between the compartments and, in the case of open systems, they also include the excretion coefficients to the environment from the different compartments. The relationship between the formulae derived and the particular connection properties of the compartments is discussed. Finally, some examples have been solved.

Algorithms↗

PubMed2002

Method for the determination of molar absorptivities of thiol adducts formed from diphenolic substrates of polyphenol oxidase.

Metabolic thiols such as cysteine and glutathione are involved in the biosynthetic pathway of phaeomelanins. They attack the o-quinones generated by polyphenol oxidase in its action on mono and o-diphenols and thus generate adducts. Determination of the molar absorptivities of these adducts is useful for spectrophotometric studies of phaeomelanin biosynthesis, antibrowning reagents in plants, and polyphenol oxidase assay methods. For their calculation, a method based on the depletion of o-diphenol by the action of polyphenol oxidase in the presence of thiol has been proposed. However, the method is slow and presents certain problems, for which reason we propose a new and faster method based on the action of polyphenol oxidase on o-diphenols which are in excess with respect to oxygen. Under these assay conditions there is rapid enzymatic formation of o-quinones, which react stoichiometrically with a thiol giving rise to the corresponding thiol-diphenol adduct. The method has been successfully applied to adducts of cysteine and glutathione with several o-diphenolic substrates of polyphenol oxidase involved in phaeomelanin biosynthesis in skin, neurones, and plants.

Agaricales↗

PubMed2002

Kinetic analysis of the general modifier mechanism of Botts and Morales involving a suicide substrate.

Suicide substrates are widely used in enzymology for studying enzyme mechanisms and designing potential drugs. The presence of a reversible modifier decreases or increases the rate of substrate-induced inactivation, with evident physiological and experimental consequences. To date, only the action of a competitive or uncompetitive inhibitor of an enzyme system involving suicide substrate has been reported. In this paper, we analyse the kinetics of enzyme-catalysed reactions which evolve in accordance with the general modifier mechanisms of Botts and Morales in which enzyme inactivation is induced by suicide substrate. Rapid equilibrium of all of the reversible reaction steps involved is assumed and the time course equations for the residual enzyme activity, the inactive enzyme forms and the reaction product are derived. Partition ratios giving the relative weight of the product and inactive enzyme concentrations, and the relative contribution to the product formation of each of the unmodified and modified catalytic routes, are studied. New indices pointing to the conditions under which the modifier acts as inhibitor or as activator are suggested. The goodness of the analytical solutions is tested by comparison with the simulated curves obtained by numerical integration. An experimental design and kinetic data analysis to evaluate the kinetic parameters from the time progress curves of the product are proposed. From these results, those corresponding to several reaction mechanisms involving both a suicide substrate and a modifier, and which can be regarded as particular cases of the general case analysed here, can be directly and easily derived.

Animals↗

PubMed2002

Unification for the expression of the monophenolase and diphenolase activities of tyrosinase.

In order to unify and generalize, we define the International Units used to express the monophenolase and diphenolase activity of mushroom tyrosinase acting on different monophenol/diphenol pairs and establish a quantitative relation. Similarly, the activity units to express tyrosinase activity proposed by suppliers are discussed and compared with the above International Units. Lastly, we study the relation between International Units of diphenolase activity and of monophenolase activity for other biological sources of tyrosinase.

Catalysis↗

PubMed2001

Mechanism of reaction of hydrogen peroxide with horseradish peroxidase: identification of intermediates in the catalytic cycle.

The mechanism of the reaction of horseradish peroxidase isoenzyme C (HRPC) with hydrogen peroxide to form the reactive enzyme intermediate compound I has been studied using electronic absorbance, rapid-scan stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies at both acid and basic pH. The roles of the active site residues His42 and Arg38 in controlling heterolytic cleavage of the H(2)O(2) oxygen-oxygen bond have been probed with site-directed mutant enzymes His42 --> Leu (H42L), Arg38 --> Leu (R38L), and Arg38 --> Gly (R38G). The biphasic reaction kinetics of H42L with H(2)O(2) suggested the presence of an intermediate species and, at acid pH, a reversible second step, probably due to a neutral enzyme-H(2)O(2) complex and the ferric-peroxoanion-containing compound 0. EPR also indicated the formation of a protein radical situated more than approximately 10 A from the heme iron. The stoichiometry of the reaction of the H42L/H(2)O(2) reaction product and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) was concentration dependent and fell from a value of 2 to 1 above 0.7 mM ABTS. These data can be explained if H(2)O(2) undergoes homolytic cleavage in H42L. The apparent rate of compound I formation by H42L, while low, was pH independent in contrast to wild-type HRPC where the rate falls at acid pH, indicating the involvement of an ionizable group with pK(a) approximately 4. In R38L and R38G, the apparent pK(a) was shifted to approximately 8 but there is no evidence that homolytic cleavage of H(2)O(2) occurs. These data suggest that His42 acts initially as a proton acceptor (base catalyst) and then as a donor (acid catalyst) at neutral pH and predict the observed slower rate and lower efficiency of heterolytic cleavage observed at acid pH. Arg38 is influential in lowering the pK(a) of His42 and additionally in aligning H(2)O(2) in the active site, but it does not play a direct role in proton transfer.

Arginine↗

PubMed2001

Catalase-like oxygen production by horseradish peroxidase must predominantly be an enzyme-catalyzed reaction.

When hydrogen peroxide (H2O2) was provided as the only substrate for horseradish peroxidase C (HRP-C) the catalase-like emission of oxygen gas was observed. The reaction was favored at neutral compared to acidic pH. Addition of the superoxide radical scavengers tetranitromethane (TNM) or superoxide dismutase (SOD) increased activity. TNM's effect was concentration dependent but SOD's was not, indicating that only some of the superoxide generated was released into solution. Manganous ions (Mn2+) react with superoxide radicals to regenerate H2O2 but not oxygen; when added to the reaction medium oxygen production was reduced but not abolished. The effect was essentially concentration independent, suggesting that most oxygen was produced enzymatically and not by chemical disproportionation of superoxide. The catalase-like activities of some site-directed mutants of HRP-C suggest that active site residues histidine 42 and arginine 38 are influential in determining this activity. A clear correlation also existed between catalase activity and the enzymes' resistance to inactivation by H2O2. Computer simulation of a reaction scheme that included catalase-like activity agreed well with experimental data.

Arginine↗

PubMed2001

Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol.

Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a monophenol (4-tert-butylphenol) has been demonstrated for the first time in the literature. The findings confirm the previously proposed mechanism to explain the action of tyrosinase on monophenols (J.N. Rodríguez-López, J. Tudela, R. Varón, F. García-Carmona, F. García-Cánovas, J. Biol. Chem. 267 (1992)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols into diphenols, giving rise to an enzyme-substrate complex in the process. The o-diphenol formed is then released from the enzyme-substrate complex or oxidized to the corresponding o-quinone. In order to detect the enzymatic release of o-diphenol, the non-enzymatic evolution of the o-quinone to generate o-diphenol by weak nucleophilic attack reactions and subsequent oxidation-reduction was blocked by the nucleophilic attack of an excess of cysteine. Furthermore, the addition of catalytic quantities of an auxiliary o-diphenol (e.g. catechol) considerably increases the accumulation of 4-tert-butylcatechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-tert-butylcatechol complex and 4-tert-butylcatechol is then released (with k(-2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) and the 4-tert-butylcatechol generated by the enzyme then enter into competition. When [catechol] >> [4-tert-butylcatechol], the enzyme preferentially binds with the catechol to close the catalytic cycle, while 4-tert-butylcatechol is accumulated in the medium. In conclusion, we demonstrate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an auxiliary o-diphenol such as catechol.

Agaricales↗

PubMed2001

Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones.

Tyrosinase can act on monophenols because of the mixture of met- (E(m)) and oxy-tyrosinase (E(ox)) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since 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 L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D(0)](ss). Hence, the system reaches an initial pseudo-steady state when t-->0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D(f)](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E(m)D, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E(ox) on monophenols, E(m)D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D(f)](ss)) necessary for the final steady state to be reached after the lag period.

Agaricales↗

PubMed2001

Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2.

H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K(2) (affinity of the first intermediate, compound I, for H2O2) and k(3) (apparent rate constant controlling catalase activity) of 4.0 +/- 0.6 mM and 1.78 +/- 0.12 s(-1) respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation.

Catalase↗

PubMed2001

The inactivation of horseradish peroxidase isoenzyme A2 by hydrogen peroxide: an example of partial resistance due to the formation of a stable enzyme intermediate.

The inactivation of horseradish peroxidase A2 (HRP-A2) with H2O2 as the sole substrate has been studied. In incubation experiments it was found that the fall in HRP-A2 activity was non-linearly dependent on H2O2 concentrations and that a maximum level of inactivation of approximately 80% (i.e. approximately 20% residual activity) was obtained with 2,000 or more equivalents of H2O2. Further inactivation was only induced at much higher H2O2 concentrations. Spectral changes during incubations of up to 5 days showed the presence of a compound III-like species whose abundance was correlated to the level of resistance observed. Inactivation was pH dependent, the enzyme being much more sensitive under acid conditions. A partition ratio (r1 approximately equals 1,140 at pH 6.5) between inactivation and catalysis was calculated from the data. The kinetics of inactivation followed single exponential time curves and were H2O2 concentration dependent. The apparent maximum rate constant of inactivation was lambdamax=3.56+/-0.07x10(-4)s(-1) and the H2O2 concentration required to give lambdamax/2 was K2=9.94+/-0.52 mM. The relationship lambdamax<ki has been shown to apply and thus the rate constant of inactivation has been calculated as ki=1.9x10(-3)s(-1). HRP-A2 possessed catalase-like oxygen gas-releasing activity, the catalytic constant being k3=2.2 s(-1), and the affinity for H2O2 as K2=23 mM. Catalase-like activity was pH dependent and favoured under more basic conditions. A mechanistic model has been developed and used to explain the behaviour of HRP-A2. The model suggests that, in common with HRP-C, mechanism-based (suicide) inactivation is being observed but that a fraction of the HRP-A2 is protected from inactivation in the form of a modified compound III species.

Catalase↗

PubMed2001

Time course equations of the amount of substance in a linear compartmental system and their computerized derivation.

In this contribution, we present the symbolic time course equations corresponding to a general model of a linear compartmental system, closed or open, with or without traps and with zero input. The steady state equations are obtained easily from the transient phase equations by setting the time --> infinity. Special attention has been given to the open systems, for which an exhaustive kinetic analysis has been developed to obtain important properties. Besides, the results have been particularized to open systems without traps and an alternative expression for the distribution function of exit times has been provided. We have implemented a versatile computer program, that is easy to use and with a user-friendly format of the input of data and the output of results. This computer program allows the user to obtain all the information necessary to derive the symbolic time course equations for closed or open systems as well as for the derivation of the distribution function of exit times.

Computer Simulation↗

PubMed2001

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible, uncompetitive inhibitor.

We present a general kinetic analysis of enzyme catalyzed reactions evolving according to a Michaelis-Menten mechanism, in which an uncompetitive, reversible inhibitor acts. Simultaneously, enzyme inactivation is induced by an unstable suicide substrate, i.e. it is a Michaelis-Menten mechanism with double inhibition: one originating from the substrate and another originating from the reversible inhibitor. Rapid equilibrium of the reversible reaction steps involved is assumed and the time course equations for the reaction product have been derived under the assumption of limiting enzyme. The goodness of the analytical solutions has been tested by comparison with simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested.

Enzyme Inhibitors↗