Françoise Labeyrie

Propriétés d'un noyau cytochromique b2 résultant d'une protéolyse de la l-lactate: Cytochrome c oxydoréductase de la levure

Abstract 1. 1. The tryptic hydrolysis of cytochrome b 2 (yeast l -lactate: cytochrome c oxidoreductase, EC 1.1.2.3) liberates a cytochromic polypeptide which can be separated from the other fragments by gel filtration. 2. 2. The properties of this new product, called “noyau cytochromique b 2 ”, have been investigated: the molecular weight is about 11 000 and this molecule is associated with one heme group; its spectral properties are very similar in the visible region to those of cytochrome b 2 . The redox potential is −0.028 V to be compared with the value 0.000 V relative to cytochrome b 2 (pH 7.00; 30°). Severa, different components have been detected by electrophoresis. These data have been used in a discussion on the structural aspects of the active molecule of lactate dehydrogenase.

Evidence by NMR for mobility of the cytochrome domain within flavocytochrome b2

According to a model proposed by Gervais, M, Groudinsky, O., Risler, Y. and Labeyrie, F. ((1977) Biochem. Biophys. Res. Commun. 77, 1543-1551) flavocytochrome b2 is composed of a central flavodehydrogenase entity of 4 X 45 kDa to which are attached four cytochrome b2 globules of approx. 11 kDa that are released after proteolysis of the connective loops. A possible inherent mobility of the latter with functional significance was suspected. Proton NMR spectra at 400 MHz of the isolated and of the flavodehydrogenase-bound ferricytochrome b2 units have been compared. In the ranges downfield of +12 ppm and upfield from -4 ppm, where hyperfine-shifted heme proton resonances reside, the chemical shifts are identical for the two forms, but the linewidths are markedly broader for flavocytochrome b2. The linewidths of three heme resonances, a methyl at +19 ppm, two single protons at -6 and -8 ppm (most probably from one vinyl) and an unassigned line at -2.4 ppm, all increase by a factor of about 4. Since, in the present case, linewidths are controlled mainly by proton/proton dipolar relaxations which are caused by molecular tumbling, a change in linewidths of about 15 would be expected if the cytochrome b2 globule had no free motion relative to the flavodehydrogenase domain. The present results thus support the previous hypothesis that such a relative mobility, of unknown correlation time and amplitude, actually exists.

d-Lacticodéshydrogénase de la levure anaérobie etude de l'inactivation irréversible par un chélateur

The kinetics of inactivation by Versene of d-lactate dehydrogenase from anaerobic yeast was studied. Inactivation is irreversible: a diminution of free Versene concentration down to 10−14M does not lead to any reactivation. The initial velocity of inactivation is proportional to Versene concentrations and to enzyme concentrations. The Michaelis constant (in the reaction d-lactate to pyruvate) of the partially inactivated enzyme is not modified. Activation energy of inactivation is low (⩽ 3kcal/mole). d-Lactate (but not l-) protects the enzyme from inactivation by decreasing the velocity of inactivation. Neither the acceptors nor flavine-adenine dinucleotide protect. Variation of initial inactivation velocity in relation to d-lactate concentration during the inactivation follows a hyperbolic kinetics: d-lactate concentration giving half protection is 7 · 10−6M, that is 300 times smaller than the Michaelis constant. Inactivation of d-lactate dehydrogenase by Versene is therefore due to the loss of a metal ion that is necessary for enzyme activity. A hypothesis is advanced that the fixation of the substrate takes place in the vicinity of the metal.

l-Lactate cytochrome c reductase: Rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly

This study is part of a series aimed at the characterization of individual steps of electron transfer taking place between prosthetic flavin, heme b2, heme c within active sites and complexes. After rapid mixing of ferricytochrome c with partially reduced flavocytochrome b2, the reaction is followed at the level of two reactants, cytochrome b2 and cytochrome c. In order to define the proper reactivity of flavosemiquinone, conditions under which this form is highly stabilized (presence of pyruvate) have been chosen. With the help of simulations, it has been possible to characterize a rapid step of electron transfer from cytochrome b2 to cytochrome c within a complex (at approx. 70% saturation) and a slow step k = 5 s-1 assigned to cytochrome b2 reduction by flavosemiquinone within the active site of the pyruvate-liganded enzyme.

Modifications of redox equilibria with semiquinone stabilization upon pyruvate binding to L-lactate cytochrome c oxidoreductase (flavocytochrome b2)

Spectral redox titrations of flavin and cytochrome b2 moieties of flavocytochrome b2 were achieved in the absence and in the presence of pyruvate under equilibrium conditions at 18 degrees C; direct measurements of spin flavosemiquinone proportions have been carried out by EPR determinations at the same temperature. Our results show that the equilibria involving flavin are largely affected by the presence of pyruvate; the semiquinone proportion markedly increases almost till unit near half-reduction of cytochrome b2; at 10 mM pyruvate, the dismutation constant, Kdism = (Fs)2/(Fo)*(Fr) increases by a factor greater than or equal to 10.

Etude cinétique de l'action de la trypsine sur la β-lactoglobuline native ou dénaturée par la chaleur

The mechanism constants and the maximum rates of reaction have been compared at various temperatures between 20 and 45°C for the reaction comprising the hydrolysis of native lactoglobulin by trypsin. The comparisons have also been made for two other forms of lactoglobulin (“s” and “f”) denatured by heat. It was found that for the native substrate, KmN is equal to 0.8 · 10−4M and does not vary appreciably with the temperature. However, for the two denatured forms, the Michaelis constant (calculated with the molecular weight of the native form) is three times as small as that for the native form, and in addition is practically independent of temperature. The relative values for the rate constant of the decomposition of the enzyme-substrate complex change with the temperature. The energies of activation are respectively 18,600 cal/mol. for the hydrolysis of native lactoglobulin and 14,200 cal/mol. for that of the denatured forms. Using these data, the variation of entropy and enthalpy of activation were calculated according to the absolute rate theory and the values found were respectively ΔS∗ = −4.5 cal/degree and ΔH∗ = + 18,000 cal/mol. for the native substrate, ΔS∗ = −18 cal/degree and ΔH∗ = + 13,600 cal/mol. for the denatured substrates. The interpretations which one can give to these results taking into consideration the complexity of proteolytic reactions, are discussed. The results appear to favour the idea that the limiting process of the overall rate corresponds in the native protein to a simultaneous breaking of a peptide linkage and of three out of four hydrogen bonds. These four hydrogen bonds, situated on both sides of the CONH bond, unite the upper part of the helix with the lower one.

Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (l-lactate cytochrome c reductase) upon binding of pyruvate, the reaction product

Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b2 moiety were carried out at room temperature, in the presence of added pyruvate or in its absence. Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate, the reaction product in the oxidation of L-lactate. The proportion of flavin semiquinone nearly reaches then 100 per cent.

Nouvelle détermination du potentiel d'oxydoréduction du système lactate-pyruvate

Abstract For the redox potential, measured in the presence of riboflavine and the D -lactic dehydrogenase of yeast, for the equilibrium D -lactate ⇌ pyruvateat pH 7 and 27°, the value of E ′ o = −0.190 V was found. This value is in good agreement with the one estimated by Burton and Wilson . It differs markedly from the earlier values determined without considering the specificity of the enzymes.

Inhibition de l'hydrolyse trypsique de la sérum albumine par les sels de sodium: Rôle de l'anion

All sodium salts, when added to a dialysed solution of serumalbumin, behave as inhibitors of tryptic proteolysis of this substrate. The concentration of salt giving 50% inhibition is 6 · 10−2M for NaCl and varies with the nature and valency of the anion in the following order: Cl− ∼ Br− ∼ CH3CO2− > SCN− > SO4−− ∼ HPO4−− ∼ C2O4−− > citrate−3 > P2O7(−3,−4) > methylorange−. The parts corresponding to the effect of ionic strength and to the fixation of the anions on the serumalbumin are discussed for the different inhibitors.

d-Lacticodéshydrogénase de la levure anaérobie III. Inactivation par la quinacrine, protection par le substrat et la flavine adénine dinucléotide

Abstract Kinetic studies of the progressive inactivation of d -lactic dehydrogenase by quinaerine, show the reaction to be first order with respect to quinacrine and enzyme and second order with respect to time. The activation energy, very small at 0° (3 kcal/mole) increases with temperature (20 kcal/mole at 20°). Elimination of free quinacrine does not bring forward any reactivation even if FAD or Zn 2+ are then added, both of them being considered as dissociable prosthetic groups. The inactivation rate is decreased if d -lactate, oxalate, a competitive inhibitor, or FAD, are added to the native enzyme. At concentrations corresponding to the saturation level of the protector, d -lactate protects 75% and FAD 98%. These experiments show an interaction and hence suggest a proximity between the substrate fixed on its receptor site and the receptor site of the flavin.