Robert Eisenthal

Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions

Xanthine oxidoreductase (XOR) catalyses the reduction of the therapeutic organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), as well as inorganic nitrate and nitrite, to nitric oxide (NO) under hypoxic conditions in the presence of NADH. Generation of nitric oxide is not detectable under normoxic conditions and is inhibited by the molybdenum site‐specific inhibitors, oxypurinol and (−)BOF 4272. These enzymic reactions provide a mechanism for generation of NO under hypoxic conditions where nitric oxide synthase does not function, suggesting a vasodilatory role in ischaemia.

Estimation of Michaelis constant and maximum velocity from the direct linear plot

When estimates of Michaelis-Menten parameters are obtained from kinetic observations taken in pairs, as in the direct linear plot, bias can arise in the final estimates if any pairs lead to negative values of the maximum velocity V. This bias can be removed by treating such negative values as if they were large and positive, and by treating the corresponding values of Km in the same way.

Antimicrobial Properties of Milk: Dependence on Presence of Xanthine Oxidase and Nitrite

ABSTRACT Human and bovine milk inhibited the metabolic activity of Escherichia coli, as shown by luminescence monitoring of constructs expressing the luxCDABE genes. Inhibition was dependent on both xanthine oxidase (XO) activity and on the presence of nitrite, implying that XO-generated nitric oxide functions as an antibacterial agent.

The dependence of enzyme activity on temperature: determination and validation of parameters

Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by DeltaG(Dagger)(cat), and irreversible inactivation defined by DeltaG(Dagger)(inact). However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E(inact)) that is in reversible equilibrium with the active form (E(act)); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, DeltaH(eq), and a new thermal parameter, T(eq), which is the temperature at which the concentrations of E(act) and E(inact) are equal; T(eq) may therefore be regarded as the thermal equivalent of K(m). Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T(eq) and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.

Specificity and kinetics of hexose transport in Trypanosoma brucei

Transport of 6-deoxy-D-glucose was studied in Trypanosoma brucei in order to characterise the kinetics of hexose transport in this organism using a nonphosphorylated sugar. Kinetic parameters for efflux and entry, measured using zero-trans and equilibrium exchange protocols, indicate that the transporter is probably kinetically symmetrical. Comparison of the kinetic constants of D-glucose metabolism with those for 6-deoxy-D-glucose transport shows that transport across the plasma membrane is likely to be the rate-limiting step of glucose utilisation. The transport rate is nevertheless very fast and 6-deoxy-D-glucose, at concentrations below Km, enters the cells with a half filling time of less than 2 s at 20 degrees C. Thus the high metabolic capacity of these organisms is matched by a high transport rate. The structural requirements for the trypanosome hexose transporter were explored by measuring inhibition constants (Ki) for a range of D-glucose analogues including fluoro and deoxy sugars as well as epimeric hexoses. The relative affinities shown by these analogues indicated H-bonds from the carrier to the C-3, C-4 and C-5 hydroxyl oxygens and from the C-1 and C-3 hydroxyl hydrogens to the binding site. Hydrophobic interactions are likely at the C-2 and C-6 regions of the glucose molecule. Spatial constraints appear to occur around C-4 indicating that the transport site at this position is not freely open to the external solution as is the case with the mammalian hexose transporter. However, the trypanosome transporter appears to accept D-fructose but the common mammalian (erythrocyte type) hexose transporter does not.

A new route to peroxynitrite: a role for xanthine oxidoreductase

Peroxynitrite, a potent oxidising, nitrating and hydroxylating agent, results from the reaction of nitric oxide with superoxide. We show that peroxynitrite can be produced by the action of a single enzyme, xanthine oxidoreductase (XOR), in the presence of inorganic nitrite, molecular oxygen and a reducing agent, such as pterin. The effects of oxygen concentration on peroxynitrite production have been examined. The physiologically predominant dehydrogenase form of the enzyme is more effective than the oxidase form under aerobic conditions. It is proposed that XOR‐derived peroxynitrite fulfils a bactericidal role in milk and in the digestive tract.

Prospects for Antiparasitic Drugs THE CASE OF TRYPANOSOMA BRUCEI, THE CAUSATIVE AGENT OF AFRICAN SLEEPING SICKNESS*

Glycolysis in the bloodstream form ofTrypanosoma brucei provides a convenient context for studying the prospects for using enzyme inhibitors as antiparasitic drugs. As the recently developed model of this system (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207–3215) contains 20 enzyme-catalyzed reactions or transport steps, there are apparently numerous potential targets for drugs. However, as most flux control resides in the glucose-transport step, this is the only step for which inhibition can be expected to produce large effects on flux, and in the computer model such effects prove to be surprisingly small (although larger than those obtained by inhibiting any other step). It follows that there is little prospect of killing trypanosomes by depressing their glycolysis to a level incapable of sustaining life. The alternative is to use inhibition to increase the concentration of a metabolite sufficiently to interfere with the viability of the organism. For this purpose, only uncompetitive inhibition of pyruvate export proves effective in the model; in all other cases studied, the effects on metabolite concentrations are little more than trivial. This observation can be explained by the fact that nearly all of the metabolite concentrations in the system are held within relatively narrow ranges by stoichiometric constraints.

The effect of temperature on enzyme activity: new insights and their implications

The two established thermal properties of enzymes are their activation energy and their thermal stability. Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive transition, in addition to an irreversible thermal inactivation. Two new thermal parameters, Teq and ΔHeq, describe the active–inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We review here the Model itself, methods for the determination of Teq and ΔHeq, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

Reduction of organic nitrates catalysed by xanthine oxidoreductase under anaerobic conditions

Xanthine oxidoreductase catalyses the anaerobic reduction of glyceryl trinitrate (GTN), isosorbide dinitrate and isosorbide mononitrate to inorganic nitrite using xanthine or NADH as reducing substrates. Reduction rates are much faster with xanthine as reducing substrate than with NADH. In the presence of xanthine, urate is produced in essentially 1:1 stoichiometric ratio with inorganic nitrite, further reduction of which is relatively slow. Organic nitrates were shown to interact with the FAD site of the enzyme. In the course of reduction of GTN, xanthine oxidoreductase was progressively inactivated by conversion to its desulpho form. It is proposed that xanthine oxidoreductase is one of several flavoenzymes that catalyse the conversion of organic nitrate to inorganic nitrite in vivo. Evidence for its further involvement in reduction of the resulting nitrite to nitric oxide is discussed.

Estimation of parameters for cell-surface interactions : Maximum binding force and detachment constant

A convenient model is presented which can be used to quantify the relationship between applied shear and attached cell fraction in cell/surface interaction studies. The model uses two parameters (the shear stress required to detach the total attached cell population and a detachment constant) based on the estimated strength of the cell/bead interaction force. Use of these parameters allows results obtained on different systems to be compared. The model has been applied to data from three systems. 1) The effects of shear on the interaction between anti-goat IgG-coated beads and surface immobilized goat IgG;1 2) The effect of applying fluid shear stress to a stable fraction of attached 3T3 fibroblast cells on glass;2 and 3) The interaction of suspended yeast cells with surface-immobilized concanavalin A which is reported here. In the yeast system, the model provided a convenient aid for quantifying the effect of competing glucose on the interaction strength where it was found that the detachment constant for yeast interaction with surface-bound conA increases with the glucose concentration while the maximum shear stress and the binding force between the yeast cells and conA decreases.