Véronique Larreta-Garde

ADDITIONAL DATA ABOUT THERMOLYSIN SPECIFICITY IN BUFFER- AND GLYCEROL-CONTAINING MEDIA

Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example in inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.

Modulation of protease specificity by a change in the enzyme microenvironment Selectivity modification on a model substrate, purified soluble proteins and gluten

Subtilisin BPN activity on a synthetic substrate is found to decrease with the concentration of soluble additives such as sugars and polyols, the catalytic efficiency of the enzyme being related to the water activity in the reaction medium. Limited hydrolysis of B chain of insulin is followed and the cleavage priority determined. When carried out in glycerol‐containing medium, both enzyme catalytic behavior and specificity are perturbed: a different cleavage order and a selectivity restriction are observed. The experiments were generalised to purified proteins and to an insoluble protein complex. The hydrolysis kinetics of purified gliadins by pepsin and of gluten by a Bacillus neutral protease are modulated in presence of water activity depressors. Glycerol is able to increase both pepsin efficiency and gluten protein solubility. The hydrolysis order is affected by water‐structuring molecules in the enzyme microenvironment and new peptides appear whatever the size and initial solubility of the substrate.

Excess substrate inhibition of soybean lipoxygenase‐1 is mainly oxygen‐dependent

Soybean lipoxygenase‐1 kinetics are known to show product and substrate inhibition. With linoleic acid as the substrate and using a simple Michaelis‐Menten formulation, we have shown that K ss, the substrate inhibition constant was increased by more than five‐fold when initial oxygen concentration was increased from 228 to 1140 μM. Excess substrate inhibition is in fact almost avoided at high initial oxygen concentration. This modification seems correlated with enzyme saturation with oxygen relative to linoleic acid, as reflected by alterations of the substrate conversion rate. Possible implications for the enzyme kinetics are discussed.

Comparison of polarographic and chemical measurements of oxygen uptake in complex media: the example of lipoxygenase reaction.

A polarographic method using a Clark oxygen electrode was used to assess oxygen concentrations in model and complex media before and during the lipoxygenase-catalyzed oxidation of linoleic acid to hydroperoxides. The results were in good agreement with those obtained with the chemical determination of dissolved oxygen. The electrode correctly responded when the dissolved oxygen concentration was decreased by the addition of water activity depressors (sorbitol, sucrose, glucose). The influence of the medium components on the gas solubility was discussed. The linear relationship between partial pressure of oxygen (determined by polarographic method) and the oxygen concentration (determined by chemical method) indicated that the Clark oxygen electrode can be used to study enzyme reactions consuming or evolving oxygen in non-Newtonian media.

Influence of polyol additive on enzyme catalytic selectivity towards different substrates

Sorbitol, added as a depressor of water activity in the reaction medium of yADH, can modify the kinetic behaviour of the enzyme towards the four substrates tested: ethanol, propanol, butanol and pentanol, as well as towards the coenzyme NAD. All apparent Km values of the alcohol substrates and NAD decreased as the additive concentration increased. However, the additive-caused modifications of the enzyme activity were found to depend on the carbon-chain length of the alcohol substrate, in other words, the catalytic selectivity of the enzyme towards different substrates was changed by the additive. These results and supplementary experiments suggested that sorbitol may have two opposing effects on the enzyme: the positive effect leads the enzyme to adopt a conformation which is more accessible to its substrates; while the negative effect results in a diffusional constraint for the enzyme reaction. Observed results were the combination of the two opposing effects.

Effect of polyhydroxylic additives on the catalytic activity of thermolysin

Abstract The catalytic activity of thermolysin during the hydrolysis of n -(3-[2-furyl]acryloyl)-Gly-Leu amide is noticeably enhanced in the presence of sugars and polyols. A series of polyhydroxylic additives were tested and the degree of activation was found to depend on both the concentration and nature of the additive. Sucrose and trehalose were found to yield the higher activation effect whereas glycerol was found to yield an inhibition of thermolysin in a large domain of concentration. A mechanism of activation based on the lowering of the energy barrier for the enzymatic reaction is proposed. This free energy barrier lowering is very likely due to water structure modification by the additives.

Orientation of Enzyme Catalysis and Specificity by Water‐Soluble Additives

Molecular interactions between an enzyme and its substrate result from a complex and still unclear interrelation between various interactions whose equilibrium determines specificity.’ Both in the cell and under industrial conditions, the biocatalysts react in heterogeneous media; the relative importance of the diverse forces regulating enzyme behavior is thus modulated, resulting in modifications on activity,2 ~tability,~ nature of the reaction ~ a t a l y z e d ~ . ~ and also on specificity of the concerned enzymes.6~~ To mimic biological media8 and try to understand the related enzymatic mechanisms, aqueous media with restricted water content have been used. Little has been done about the influence of water on enzyme specificity when the biocatalyst reacts in an aqueous medium, though it has been shown that bound water molecules play a determining role on affinity and specificity.y Water activity was decreased by addition of polyols at high concentrations to the reaction media. In such water-limited media, the usual reaction scheme “one substrate, one enzyme, one product” is no longer true and an orientation of the enzyme selectivity may be observed. The interaction between the nature of the reaction medium and the enzyme catalysis has been studied with two different types of enzymes, allowing the recovery of various products from a single substrate, depending on the expressed enzyme selectivity. First, proteases were used, as the action of the enzyme on a known peptide or protein may give rise to various products depending on the cleavage priority expressed by the biocatalyst. Then, soybean lipoxygenase was chosen, as the enzyme may oxidize linoleic acid either on carbon 9 or on carbon 13 allowing the recovery of different hydroperoxides and derivatives.

[40] Immobilized organelles in cross-linked proteins

Publisher Summary Knowledge concerning cell organelles, such as mitochondria and chloroplasts, is now considerable; this permits the studies of structure–function relationships, particularly those involved in vectorial metabolism, energy transduction, and electron-transport processes. Organelle immobilization avoids extraction and purification phases and allows multistep reactions. Immobilization increases organelle longevity and permits their use in bioconversion processes, such as adenosine triphosphate (ATP) regeneration, photoproduction of hydrogen gas, and metabolite oxidation. This chapter discusses the preparation and stabilization of such immobilized organelles. Concerning the photosystems, the preservation of activity has been studied for conditions under which they are functionally dormant or active. An understanding of the mechanical properties and ultrastructural preservation of an immobilized organelle system is important to properly predict its performance during continuous use. Therefore, spectroscopic techniques yielding information on the state of chlorophyll–protein complexes and energy transfer should be combined with the results of fine structural and biochemical investigations.

Comparison of Macromolecular and Molecular Effects of Cosolvents on the Catalytic Behaviour of Soybean Lipoxygenase-1

The addition of sugars and polyols to the reaction medium of soybean lipoxygenase-1 confers it properties analogous to those of cytoplasm and can modulate the catalytic behaviour of the enzyme. In such non conventional media, changes can be observed at two different levels: additives exert macroscopic effects on physico-chemical and/or thermodynamic parameters of the reaction medium i.e., water activity, viscosity, surface tension; cosolvents may also exert effects at the molecular level by directly modifying enzyme conformation. Both effects may affect enzyme catalysis, and their relative contribution to the modulation of lipoxygenase-1 activity and specificity has been evaluated. When additive concentrations were higher than a threshold of around 35g additive/100g solution, macromolecular effects limited enzyme catalysis as shown by a diffusion-reaction modelling. Below this value, slight enzyme structural changes were responsible for the observed variations in lipoxygenase behaviour, including activation.

Proteolysis of Aggregated Fibronectin A Model for In Vivo Matrix Degradation

The extracellular matrix (ECM) is a network of several proteins representing both a barrier delimiting the tissues and a substratum for cell adhesion, migration, and differentiation.1 Its degradation by mammalian Zn2+-proteinases referred to as MMPs (for matrix metalloproteinases) is implied in many normal or pathological processes (development, inflammation, metastasis, dissemination, etc.).2 Fibronectin (FN) is a major structural and functional protein of the ECM. It is a dimeric protein mainly composed of three different types of homologous modules that are grouped in compact domains interconnected by flexible strands.3 Each domain presents numerous binding sites (cell binding domain, collagen, heparin, DNA binding domains, etc.) (see FIGURE 1, upper). FN modules are highly resistant to proteolysis, but observable cleaved sites are located on the connecting strands. Furthermore, many FN proteolytic fragments have been shown to present functions that are not observed in the intact protein.4 Precise knowledge of the in vivo mechanisms of FN fragment production is thus of high interest. This study presents an original approach to the kinetics of multiple cleavage site proteolysis of large proteins such as FN (Mr, 500 × 103). We have determined the proteolytic cascade leading to apparition of different FN fragments and the velocity at which cleavages occur, using thermolysin as a model for MMPs. Proteolysis has been studied using FN under two forms: as a soluble form (as found in plasma) and as a reticulated insoluble form that mimics the aggregated form that is incorporated into ECM. Hence, contrary to the usual studies dealing with enzymology under heterogeneous conditions, the substrate is here immobilized and the soluble enzyme diffuses into it.