Jean Buc

Enzymatic and physiological properties of the tungsten‐substituted molybdenum TMAO reductase from Escherichia coli

The trimethylamine N‐oxide (TMAO) reductase of Escherichia coli is a molybdoenzyme that catalyses the reduction of the TMAO to trimethylamine (TMA) with a redox potential of + 130 mV. We have successfully substituted the molybdenum with tungsten and obtained an active tungsto‐TMAO reductase. Kinetic studies revealed that the catalytic efficiency of the tungsto‐substituted TMAO reductase (W‐TorA) was increased significantly (twofold), although a decrease of about 50% in its kcat was found compared with the molybdo‐TMAO reductase (Mo‐TorA). W‐TorA is more sensitive to high pH, is less sensitive to high NaCl concentration and is more heat resistant than Mo‐TorA. Most importantly, the W‐TorA becomes capable of reducing sulphoxides and supports the anaerobic growth of a bacterial host on these substrates. The evolutionary implication and mechanistic significance of the tungsten substitution are discussed.

Purification and characterization of wheat germ hexokinases

During the last few years much work has been performed on the structure of yeast hexokinases [l-8]. Evidence obtained for a subunit structure of these enzymes shows that hexokinases exist as a tetramer [ 1, 2, 4, 5, 71, or as a dimer [8] , of molecular weight approximately 98,000, which can dissociate into two subunits (molecular weight about 49,000). Equilibrium dialysis measurements [9] have shown that each subunit is able to bind one molecule of glucose. However, our knowledge concerning the hexokinases of higher plants is still in its infancy. Until now, only crude preparations have been obtained [lo] The aim of the present work was to purify, for the first time, wheat germ hexokinases and to determine some of their physico-chemical characteristics.

Kinetic Studies of a Soluble αβ Complex of Nitrate Reductase A from Escherichia Coli

A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.

Lipolysis and heterogeneous catalysis. A new concept for expressing the substrate concentration

A new concept is proposed for quantifying the substrate concentration during heterogeneous catalysis of the kind which occurs during lipolysis. The number of molecules of protein (enzyme) adsorbable to the lipid substrate interface per unit of volume was evaluated and defined as a volumetric concentration of protein (enzyme) binding site (PEBS). Using porcine pancreatic lipase (EC 3.1.1.3) as a model enzyme, the maximal PEBS concentration was measured under various assay conditions by determining the saturation of the lipid substrate with the enzyme. Abacuses correlating the lipid substrate concentration (M) with the PEBS concentration (M) under each experimental conditions were used to express the kinetic data in terms of a volumetric concentration of PEBS. Comparisons could thus be made between data obtained with various enzymes and lipid interfaces because they were expressed with the same unit. In the case of pancreatic lipase, using triolein and tributyrylglycerol as substrates,Km values of 2.7 and 7.5 nM PEBS were obtained, respectively, andKD values ranging around 9 nM PEBS were also obtained from Scatchard plots. In addition, the average superficial density of PEBS was found to be 10×1011 molecules·cm−2, which is a value commonly obtained with structural proteins and enzymes adsorbed to an acylglyceride-water interface, this finding supports the idea that the PEBS concept represents the room in which the protein molecule adsorbs at the lipidic interface.

Molecular properties and thioredoxin-mediated activation of spinach chloroplastic NADP-malate dehydrogenase

There is no doubt that light-generated reducing equivalents can be used in C3 plants to perform a number of reactions occurring outside the chloroplasts [1-3]. Since the chloroplastic membrane is impermeable to NADPH and to reduced ferredoxin, it is currently believed that these reduction reactions in the cytoplasm involve the so-called C4dicarboxylic acid shuttle [4]. A specific malate translocator located on the chloroplastic membrane exports, outside the chloroplast, this dicarboxylic acid which is used in the cytosol to perform a number of reduction reactions [3]. NADPH formed during photosynthesis is used to reduce oxaloacetate to malate in the chloroplastic stroma. Chloroplastic malate dehydrogenase plays therefore an important role in the generation of reducing power used in reactions occurring in the cytoplasm. Moreover, crude extracts containing chloroplastic malate dehydrogenase are totally inactive in the dark and become active upon illumination. The effect of light is mimicked by a mixture of dithiothreitol and thioredoxin [5,6]. Surprisingly, chloroplastic NADP-malate dehydrogenase has never been purified to homogeneity. Therefore, nothing is known as to its structure, its reaction mechanism and the molecular bases of photoregulation by thioredoxin. Moreover one totally ignores whether several iso-forms of malate dehydrogenase exist in the chloroplast. The aim of this paper is to describe a purification procedure of chloroplastic NADP-malate dehydrogenase, to report some molecular properties of this enzyme as well as some features of its activation by dithiothreitol and thioredoxin.

The pH-induced dissociation of fructose 1,6-bisphosphatase of spinach chloroplasts

Fructose 1,6-bisphosphatase from higher plants is receiving considerable attention [l-4]. The reason for this interest rests on the belief that this enzyme plays a major role in the control by light of the Calvin cycle [5,6]. Buchanan and his colleagues have suggested [7,8] that thioredoxin, reduced by electrons from photosystem I, reacts with fructose 1,6-bisphosphatase, thus reducing disulfide bonds of this enzyme and leading to a conformation change itself associated with a modulation of its activity. Fructose 1 ,Gbisphosphatase from spinach chloroplasts is a tetramer with apparently identical subunits and a MW of 160 000. Raising the pH from 7.5 to 8.8 results in the dissociation of the enzyme in two equal halves of MW 80 000 121. Whereas the tetramer is active the dimer is almost devoid of activity. Since it is widely accepted that the pH of the chloroplast stroma changes upon illumination [9], it is of interest to study the kinetics of a conformational transition associated with the dissociation reaction and to relate this process to a possible change in the accessibility of sul~ydryl groups, for these groups are believed to play a key role in the activation-deactivation process of the enzyme [2]. This is the aim of the present paper.

Lipase-catalysed acylation of alcohols by fatty acid anhydrides. Evaluation of the selectivity based on kinetic measurements.

Abstract A method based on the analysis of kinetic measurements is described for evaluating enantioselectivity of the lipase-catalysed acylation of alcohols by fatty acid anhydrides in cases where enantiomer separation is not feasible.

A spectrophotometric method for kinetic studies with quinone-dependent oxidoreductases. Application to detection in membranes of nitrate reductase activity with menadione and duroquinone as electron donors

A simple method for estimating the activity of membrane-bound quinone-dependent oxidoreductases is described. Nitrate reductase from membranes of Escherichia coli is used as a model with menadione and duroquinone, commercially available analogues of the physiological substrates, menaquinone and ubiquinone, as electron donors. These analogues are reduced by KBH4 (which is specific for aldehydes and ketones) using molar ratios of [KBH4][menadione] = 20 and [KBH4][duroquinone] = 10. The appearance of the oxidized state can be monitored with a classical spectrophotometer and does not require a dual wavelength or diffusing-medium apparatus. To avoid turbidity in the cuvette, small amounts of membrane containing overexpressed enzyme are used.

Sensing of Chemical Signals by Enzymes

Living systems are able to sense the intensity of chemical signals originating from the external milieu and are able to detect whether this intensity increases or decreases. Chemotaxis of bacteria represents a striking example of these sensory properties defined at a rather simple level (Koshland, l979, 1980ab, 1981). Bound enzyme systems in which diffusion is a limiting process may also display these sensory properties (Engasser & Horvath, 1974, 1976; Ricard, 1987). This implies that the system possesses a memory and that its response is different depending on whether the concentration of a ligand increases or decreases. In other words the response of the system is sensitive to its history. Nucleic acids may display metastable secondary structures upon their titration and therefore may exhibit hysteresis effects (Revzin et al., 1973, Neumann, 1973, Schneider, 1976).

Thermodynamics and Kinetics of the Interactions of Thioredoxin Fb with Fructose Bisphosphatase from Spinach Chloroplast

One of the most important process involved in the photoregulation of chloroplast enzymes is their oxidoreductive modification by protein mediators known as thioredoxins (1). Different molecular species of thioredoxins from Spinach chloroplast have been purified and characterized (2). The reductive activation of fructose bisphosphatase is due to the reduction of two disulfide bridges followed by a conformation change of the protein (3). Very little was known, till recently about molecular interaction occuring between the enzyme and its protein effector. We discuss here the binding properties and the kinetics of inactivation of fructose bisphosphatase (FBPase) by thioredoxin fb.