PubMed Health⌕ Search

Biomedical subjects

R Varón

Publications and source records attributed to R Varón.

At least 55 records · Page 3Linked to original sources
PubMed1994

The effect of pH on the suicide inactivation of frog epidermis tyrosinase.

This paper presents a new reaction mechanism for the effect of the pH on the suicide inactivation of the diphenolase activity of tyrosinase. The applicability of the mechanism is supported by the experimental characterization of the kinetic behaviour of the frog epidermis enzyme acting on catechol, L-dopa and alpha-methyldopa at several pH values. Two enzyme froms 'met-' and 'oxy-' tyrosinase, but no their corresponding enzyme-diphenol complexes, present one ionizable group with very similar value of Ka which has been determined. The highest values of catalytic and inactivation efficiencies correspond to alpha-methyldopa and catechol, respectively. These kinetic studies have been carried out by using the transient phase approach previously developed, with negligible substrate consumption during the assay time. That illustrate the usefulness of the method for multisubstrate enzyme reactions.

Animals↗
PubMed1994

Kinetic analysis of reversible closed bicyclic enzyme cascades covering the whole course of the reaction.

A kinetic analysis of the closed bicyclic enzyme cascades is presented. 1. It includes the dependence on time from the onset of the reaction, of the concentration of the modified and unmodified enzyme species involved and the time course equations of the modificational fractions of the interconvertible enzymes. 2. The transient phase equations obtained allow the definition of new regulatory modification properties. 3. The expressions for concentrations of the unmodified and modified forms of the interconvertible enzymes, as well as those of the fractional modifications in the steady state are derived as particular cases of the general equations. 4. These steady state expressions coincide with those obtained by other authors. 5. The analytical results obtained are discussed in relation to the Escherichia coli glutamine synthetase cascade.

Enzymes↗
PubMed1994

Final phase of enzyme reactions following a Michaelis-Menten mechanisms in which the free enzyme and/or the enzyme-substrate complex are unstable.

An important kinetic analysis of unstable enzyme systems was carried out by Duggleby (Duggleby, R.G. (1986) J. Theor. Biol. 123, 67-80). This author states that his results are of general validity in the sense that the instability rate constants may have any value. Later, Wang & Tsou (Wang and Tsou (1990) J. Theor. Biol. 142, 531-549) rediscovered Duggleby's results when they analyzed a scheme in which the inactivations were due to a non-complexing irreversible inhibitor, pointing out the need to assume an initial steady-state in the catalytic route of the reaction. In the present contribution we show that there are values of the instability rate constants for which the equations of Duggleby are not applicable. We propose, for these cases, an alternative equation, which relates the final substrate concentration with the initial ones of both the substrate and the enzyme. Based on this, an experimental design for the evaluation of kinetic parameters is suggested. The present work concerns enzyme reactions evolving according to a Michaelis-Menten mechanism, in which the free enzyme and/or the enzyme-substrate complex are unstable.

Algorithms↗
PubMed1994

Kinetic analysis of the opened bicyclic enzyme cascades.

A kinetic analysis of the opened bicyclic enzyme cascade is presented. It includes the time-dependence of the concentrations of the modified and unmodified forms of the interconvertible enzymes, as well as their fractional modifications, from the onset of the reaction to its completion. The transient phase equations obtained allow the definition of new regulatory properties. The expressions corresponding to the concentrations and fractional modification in the steady-state are derived as particular cases of the general transient phase equations. These steady-state expressions agree with those obtained by other authors.

Enzymes↗
PubMed1993

A kinetic study of an unstable enzyme measured through coupling reactions. Application to the self-inactivation of detergent-solubilized Ca(2+)-ATPase from sarcoplasmic reticulum.

A methodology for the kinetic study of the self-inactivation of an unstable enzyme has been developed by using the transient-phase approach when the enzymatic activity is measured through a coupled enzyme system. An experimental design has been developed and applied to the inactivation of the Ca(2+)-ATPase from sarcoplasmic reticulum solubilized in the monomeric state. The catalytic activity of the ATP hydrolysis is determined in the presence of pyruvate kinase and lactate dehydrogenase as auxiliary enzymes, and the oxidation of the last substrate, NADH, is continuously monitored. The experimental results show that both substrates, ATP and calcium, protect against enzyme inactivation. This enzyme, the monomeric ATPase, fulfills the catalytic cycle of the native ATPase, and free enzyme and first-calcium bound enzyme are proposed as the intermediates which are being inactivated.

Adenosine Triphosphate↗
PubMed1993

Oxygen Michaelis constants for tyrosinase.

The Michaelis constant of tyrosinase for oxygen in the presence of monophenols and o-diphenols, which generate a cyclizable o-quinone, has been studied. This constant depends on the nature of the monophenol and o-diphenol and is always lower in the presence of the former than of the latter. From the mechanism proposed for tyrosinase and from its kinetic analysis [Rodríguez-López, J. N., Tudela, J., Varón, R., García-Carmona, F. and García-Cánovas, F. (1992) J. Biol. Chem. 267, 3801-3810] a quantitative ratio has been established between the Michaelis constants for oxygen in the presence of monophenols and their o-diphenols. This ratio is used for the determination of the Michaelis constant for oxygen with monophenols when its value cannot be calculated experimentally.

Basidiomycota↗
PubMed1993

Kinetic behaviour of zymogen activation processes in the presence of an inhibitor.

A global kinetic analysis of a general zymogen activation model, where not only the activating but also the activated enzyme suffer an irreversible inhibition is presented. A reaction in which the enzyme acts upon a substrate is coupled to monitor the process. In addition, we determined the corresponding kinetic equations for a number of particular cases of the general model studied. Finally, a kinetic data analysis and a procedure to discriminate among the different mechanisms considered, which are based on the kinetic equations obtained, are suggested.

Amino Acid Sequence↗
PubMed1993

The kinetics of enzyme systems involving activation of zymogens.

A general model of zymogen activation is proposed and explicit kinetic equations for the time courses of the various species and products involved are given. These equations are valid for the whole course of the reaction and therefore for both the transient phase and the steady state. This model is sufficiently general to include mechanisms possessing one or more steps of zymogen activation besides possible steps of inhibition (reversible or irreversible) or inactivation.

Enzyme Activation↗
PubMed1993

Oxidation of 6-hydroxydopamine catalyzed by tyrosinase.

1. The oxidation of 3,4-dihydroxyphenylethylamine (dopamine) by O2 catalyzed by tyrosinase yields 4-(2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaminequinone-H+), which evolves non-enzymatically through two branches or sequences of reactions, whose respective operations are determined by the pH of the medium. 2. The cyclization branch of o-dopaminequinone-H+ takes place in the entire range of pH and is the only significant branch at pH > or = 6. 3. The hydroxylation branch of o-dopaminequinone-H+ only operates significantly at pH < 6, and involves the accumulation of 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine), identified by high performance liquid chromatography (HPLC). 4. 6-hydroxydopamine is also a substrate of tyrosinase. The identification and evolution of the oxidation products of 6-hydroxydopamine has been carried out by spectrophotometry and HPLC assays.

Catalysis↗
PubMed1992

Catalytic oxidation of 2,4,5-trihydroxyphenylalanine by tyrosinase: identification and evolution of intermediates.

The oxidation of 3,4-dihydroxyphenylalanine (dopa) by O2 catalyzed by tyrosinase yields 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaquinone-H+). This evolves non-enzymatically through two branches (cyclization and/or hydroxylation), whose respective operations are determined by pH. The hydroxylation branch of o-dopaquinone-H+ only operates significantly at pH < or = 5.0 and involves the accumulation of 2,4,5-trihydroxyphenylalanine (topa), which has been detected by high-performance liquid chromatography (HPLC). This last compound is also a substrate of tyrosinase. The oxidation of topa by both tyrosinase and periodate yields 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2-benzoquinone, with its amino group protonated (o-topaquinone-H+), which is red (RTQH) (lambda max 272-485 nm) at pH 7.0 and yellow (TTQH) (lambda max 265-390 nm) at pH 3.0. This is based on pKa 4.5 of the 2-OH group of the benzene ring of o-topaquinone-H+, as derived from spectrophotometric and HPLC assays. At physiological pH, RTQH undergoes deprotonation of the ammonium group of the side chain to yields RTQ, which cyclize into 2-carboxy-2,3-dihydroxyindolen-5,6-quinone (dopachrome), with a 1:1 stoichiometry and first-order kinetics. The evolution of RTQH has been analyzed by spectrophotometry, HPLC, cyclic voltammetry and constant potential electrolytic assays. From HPLC assays, the value of the first-order constant for the evolution of RTQH at pH 7.0 (kRTQHapp 4.83 x 10(-5) s-1), as well as of the rate constant for the cyclization step of RTQ (kRTQc 2.53 x 10(-3) s-1) were determined.

Animals↗
PubMed1992

A kinetic study of the generation and decomposition of some phenothiazine free radicals formed during enzymatic oxidation of phenothiazines by peroxidase-hydrogen peroxide.

A kinetic study of the oxidation of four different phenothiazines (Pts) by peroxidase-hydrogen peroxide was carried out. The free radical formed during the enzymatic oxidation suffers a non-enzymatic breakdown and the overall system was analysed and characterized. The non-enzymatic breakdown of the cation radical does not occur through a disproportionation mechanism but through a more complex mechanism. The kinetic parameters of the overall system were determined for the different Pts. These experimental data may serve in the understanding of the pharmacological action of Pts.

Buffers↗
PubMed1992

Determination of the molar absorptivities of phenothiazine cation radicals generated by oxidation with hydrogen peroxide/peroxidase.

Phenothiazines are used as antipsychotic drugs and as reagents to determine microamounts of hemoglobin in biological fluids and tissues. Several agents cause the oxidation of phenothiazines to chromophoric cation radicals, whose stability may be related with their biological action. Enzymes and proteins with peroxidase activity catalyze the oxidation by H2O2 of phenothiazines to their corresponding cation radicals, which suffer a nonenzymatic breakdown. The instability of these cation radicals makes the determination of their respective molar absorptivities very difficult. These properties, however, have been determined for a few phenothiazine cation radicals by cumbersome or unreliable procedures. In this paper a new method is proposed and applied to six different phenothiazines oxidized with H2O2/peroxidase. The method involves the stoichiometric exhaustion of H2O2, under assay conditions which yield a fast enzymatic formation of phenothiazine cation radicals and which slow down their nonenzymatic breakdown. This method may be useful for quantitative studies on the enzymatic activity and the reaction mechanism of the oxidation of a number of phenothiazines catalyzed by different types of peroxidase, as well as by proteins with peroxidase activity, such as hemoglobin.

Algorithms↗
PubMed1992

Calibration of a Clark-Type oxygen electrode by tyrosinase-catalyzed oxidation of 4-tert-butylcatechol.

A procedure for calibrating a Clark-type oxygen electrode is described. This method is based on the oxidation of 4-tert-butylcatechol (TBC) by O2 catalyzed by tyrosinase, to yield 4-tert-butyl-o-benzoquinone (TBCQ). This reaction consumes known amounts of oxygen in accordance with the stoichiometry: 2TBC + O2----2TBCQ + 2H2O and can be used to determine the relation between the oxygen concentration and the oxygen electrode response. TBCQ is very stable in the reaction medium for more than 30 min and shows no significant breakdown, which makes the calibration possible. A kinetic study of the oxidation of 3,4-dihydroxyphenylalanine by tyrosinase using the oxygen electrode is shown to confirm the validity of the calibration method.

Calibration↗
PubMed1992

Kinetic analysis of the control through inhibition of autocatalytic zymogen activation.

A global kinetic analysis of a model of an autocatalytic zymogen-activation process in which an irreversible inhibitor competes with the zymogen for the active site of the proteinase is presented. Processes like the one here described are of great physiological interest because they are involved in the enzyme regulation of the gastrointestinal-tract enzymes, in blood coagulation, in fibrinolysis and in the complement system. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. In addition, we have introduced a new parameter related to the kinetic behaviour of the system which allows us to predict whether the inhibition route or the activation route prevails in the steady state of the system. Finally, we extend the kinetic equations derived to different particular cases of the system studied.

Catalysis↗
PubMed1992

Analysis of a kinetic model for melanin biosynthesis pathway.

The kinetic behavior of the melanin biosynthesis pathway from L-tyrosine up to dopachrome has been studied from experimental and simulation assays. The reaction mechanism proposed is based on a single active site of tyrosinase. The diphenolase and monophenolase activities of tyrosinase involve one single (oxidase) and two overlapped (hydroxylase and oxidase) catalytic cycles, respectively. The stoichiometry of the pathway implies that one molecule of tyrosinase must accomplish two turnovers in the hydroxylase cycle for each one in the oxidase cycle. Furthermore, the steady-state rates of dopachrome production and O2 consumption from tyrosine and L-dopa, also fulfill the stoichiometry of the pathway: VO2T/VDCT = 1.5 and VO2T/VDCD = 1.0, where T represents L-tyrosine, DC represents dopachrome, and D represents L-dopa. It has been ascertained by high performance liquid chromatography that in the steady-state, a quantity of dopa is accumulated ([D]ss) which fulfills the constant ratio [D]ss = R[T]0. Taking this ratio into account, an analytical expression has been deduced for the monophenolase activity of tyrosinase. In this expression kcatT congruent to (2/3)k3(K1/K2)R, revealing that kcatT is not a true catalytic constant, since it also depends on equilibrium constants and on the experimental R = 0.057. This low value explains the lower catalytic efficiency of tyrosinase on tyrosine than on dopa, (VmaxT/KmT)/(VmaxD/KmD) congruent to (2/3)R, since a significant portion of tyrosinase is scavenged from the catalytic turnover as dead-end complex EmetT in the steady-state of the monophenolase activity of tyrosinase.

Basidiomycota↗
PubMed1992

Kinetics of a model of autocatalysis, coupling of a reaction in which the enzyme acts on one of its substrates.

A global kinetic analysis is presented of a model of an enzyme autocatalytic process, to which a reaction is coupled, in which the enzyme acts upon one of its substrates. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. In addition, we determine the corresponding kinetic equations for several particular cases which are characterized by certain relations between the rate constants. Finally, a kinetic data analysis is proposed for one of these particular cases. It can easily be extended to any of the other cases.

Catalysis↗