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.

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.

Gel-sol transition can describe the proteolysis of extracellular matrix gels.

We monitored the cell-free solubilization of extracellular matrix and fibronectin gels, catalyzed by exogenous proteinases. The corresponding measurements could not be interpreted according to usual proteinase kinetics. The observation that this experimental system did not consist in surface but in bulk degradation and appeared specific to gel substrates, incited us to use gelation-related approaches to describe these kinetics. We show that the gel-sol transition theory adequately describes the enzyme reactions. This allowed formulation and experimental confirmation of a power law relating macroscopic changes of the gel to enzyme kinetics. This approach could also be used for other power laws predicted by the gel-sol transition theory, allowing a better understanding of macroscopic modification of the extracellular matrix during proteolysis, which is implied in many biological situations, especially tumor dissemination.

Glycerol's influence on the oxidized insulin B-chain conformation in relation to the selectivity variation of subtilisin: an nuclear magnetic resonance and simulated annealing study.

Glycerol, employed to mimic biological media with restricted water activity, has been shown to modify the activity of subtilisin BPN', an endopeptidase, towards the oxidized insulin B-chain, a well-studied substrate (FEBS Lett., 279 (1991) 123-131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the structure of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain tertiary structure loses its central helix (residues B9-B19) and presents a folded structure with a flexible turn (residues B18-B24) in the beta-turn region of the insulin B-chain; whereas, in glycerol, the peptide is more rigid and is not folded. Moreover, in our experimental conditions, glycerol favors beta-strand secondary structure formation. Following these results, hypotheses about the differences observed in enzymatic activity on this substrate in glycerol have been postulated.

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 microM. 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.

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 behaviour 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.

Stabilization of biological photosystems : immobilization of thylakoids and chromatophores for hydrogen production and ATP regeneration.

Lettuce thylakoïds were immobilized by the action of glutaraldehyde at subzero temperature in the presence of albumin. Foam structures with good mechanical properties were obtained. The activity yields for photosystem II and photosystems I + II were found equal to 71 per cent and 35 per cent respectively. The yield for ATP regeneration from ADP and Pi was 26 per cent. Increases of stability after immobilization were observed for all the functions of thylakoïds when stored and when continuously working. Spheroplasts and chromatophores from Rhodopseudomonas capsulata were immobilized with the same method; yields for ATP regeneration were found equal to 40 per cent and 70 per cent, respectively. An important increase of stability after immobilization was observed in both cases.

Cofactor regeneration in immobilized enzyme systems: chemical grafting of functional NAD in the active site of dehydrogenases.

One of the limiting steps in the further development of enzyme technology is the regeneration of cofactors, especially the pyridinic nucleotide cofactors. Immobilization of alcohol dehydrogenase and steroid dehydrogenase is described. In the last case stabilized enzymes could work in non aqueous solvents. Co-enzyme molecules are bound in the immediate vicinity of the active site of the enzyme. Cofactor regeneration was performed with an electron carrier (Phenazine methosulfate). Ageing phenomena were observed. The co-immobilization of superoxide dismutase gives rise to an increase of stability.

Modulation of egg-white lysozyme activity by viscosity intensifier additives.

The activity of egg-white lysozyme was measured in the presence of carbohydrate additives in the reaction medium. These additives show a significant affinity for water. They depress water activity and increase the viscosity of the medium. Solute-solvent interactions in aqueous solutions of the additives are characterized by properties such as the intrinsic viscosity, Huggins constant apparent molar volume and hydration number. It was found that, despite the lowering of enzyme activity when the concentration of additive is increased, the behavior remains Michaelian and neither modification of Km nor inhibition by excess substrate is observed. On the other hand, the effect of the viscosity of the medium on enzyme activity was determined. This effect is independent of the nature of the additive at high viscosities (greater than 4 mPa s-1) for which enzyme activity is very low and appears to vary according to the kind of additive in dilute solution at low viscosities (less than 2 mPa s-1).

Structural studies by 1H NMR and molecular modeling of peptide substrates of thermolysin in relation with its proteasic activity in water and glycerol.

In an attempt to explain the relationship between conformations of peptide substrates of thermolysin in natural form and the experimental enzymatic cleavages, five peptides of various length were studied in two solvents H2O and glycerol, which may mimic the catalytic environmental conditions. As NMR failed to define sufficiently rough constraints to ensure a convergence of a refinement process for such short and flexible peptides, the conformational space was first searched using the MCMM method. The generated structures were then clustered in families using a 0.3A rmsd criterion and the derived structural characteristics were compared to the experimental NMR parameters. In a first approach, the NMR consistent conformations were compared with the structure of a thermolysin bound peptidic inhibitor ZG(P)LL to characterize the free-ligand predisposition to be cleaved. Further molecular dynamic calculations were performed at 300 K on the conformations corresponding to families in agreement with the ZG(P)LL structure in order to obtain information on their stability and on the trajectories of the torsion angles involved in the active site recognition. In conclusion, for four studied peptides, some conformations were found to be in agreement with 5 of the 8 cleavages experimentally observed.

Fibrin-polyethylene oxide interpenetrating polymer networks: new self-supported biomaterials combining the properties of both protein gel and synthetic polymer.

Interpenetrating polymer network (IPN) architectures were conceived to improve the mechanical properties of a fibrin gel. Conditions allowing an enzymatic reaction to create one of the two networks in IPN architecture were included in the synthesis pathway. Two IPN series were carried out, starting from two polyethylene oxide (PEO) network precursors leading to different cross-linking densities of the PEO phase. The fibrin concentration varied from 5 to 20 wt.% in each series. The behavior of these materials during dehydration/hydration cycles was also studied. The mechanical properties of the resulting IPN were characterized in the wet and dry states. These self-supported biomaterials combine the properties of both a protein gel and a synthetic polymer. Finally, cells were grown on PEO/fibrin IPN, indicating that they are non-cytotoxic.

Recoil polarization for delta excitation in pion electroproduction.

We measured angular distributions of recoil-polarization response functions for neutral pion electroproduction for W = 1.23 GeV at Q(2) = 1.0 (GeV/c)(2), obtaining 14 separated response functions plus 2 Rosenbluth combinations; of these, 12 have been observed for the first time. Dynamical models do not describe quantities governed by imaginary parts of interference products well, indicating the need for adjusting magnitudes and phases for nonresonant amplitudes. We performed a nearly model-independent multipole analysis and obtained values for Re (S(1+)/M(1+)) = -(6.84 +/- 0.15)% and Re (E(1+)/M(1+)) = -(2.91 +/- 0.19)% that are distinctly different from those from the traditional Legendre analysis based upon M1+ dominance and ll(pi) < or = 1 truncation.

Oxygen concentration determines regiospecificity in soybean lipoxygenase-1 reaction via a branched kinetic scheme.

The effect of oxygen concentration on the regiospecificity of the soybean lipoxygenase-1 dioxygenation reaction was studied. At low oxygen concentrations (<5 microM), a dramatic change in the regiospecificity of the enzyme was observed with the hydroperoxy-octadecadienoic acid (HPOD) 13:9 ratio closer to 50:50 instead of the generally reported 95:5. This alteration of regiospecificity is not an isolated phenomenon, since it occurs during a reaction carried out under "classical" conditions, i.e. in a buffer saturated with air before the reaction. beta-carotene bleaching and electronic paramagnetic resonance findings provided evidence that substrate-derived free radical species are released from the enzyme. The kinetic scheme proposed by Schilstra et al. (Schilstra, M. J., Veldink, G. A. & Vliegenthart, J. F. G. (1994) Biochemistry 33, 3974-3979) was thus expanded to account for the observed variations in specificity. The equations describing the branched scheme show two different kinetic pathways: a fully enzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 95:5, and a semienzymatic one leading to a regio-isomeric composition of 13-HPOD:9-HPOD = 50:50. The ratio between the two different pathways depends on oxygen concentration, which thus determines the overall specificity of the reaction.

Modification by immobilization of the microenvironment of chromatophores of Rhodopseudomonas capsulata. The influence on light-induced ADP phosphorylation coupled to cyclic electron transport.

Rhodopseudomonas capsulata chromatophores were immobilized with a co-crosslinking method. Immobilization was used as a tool for a defined modification of the chromatophore environment to study ATP production over a long period of time. The light-induced phosphorylation of ADP as a function of time was studied with chromatophores under different conditions: (a) native chromatophores with and without the hexokinase ATP-trapping system; (b) immobilized chromatophores without hexokinase, with the enzyme added in the bulk solution and with the enzyme co-immobilized in the matrix. The overall amount of ATP produced as a function of ADP concentration was studied for native and immobilized chromatophores. The global phosphorylation performed was also studied as a function of the amount of biological material used. The results can be explained by an effect of the ATP/ADP ratio. The results given by the immobilization show that the important point is not the ATP/ADP ratio in the bulk solution but the ratio value in the microenvironment of the chromatophore itself.

Factors controlling initial and long-term ATP regeneration catalyzed by immobilized chromatophores.

Photosynthetic ATP regeneration was measured in open reactors using immobilized Chromatophores from Rhodopseudomonas capsulata. The influence of several factors on both initial and long-term ADP photophosphorylation was studied. The effect of phosphate salts and of bovine serum albumin on the organelle activity yield was studied. Photophosphorylation was initiated either with ADP or regenerated ATP and the roles of these nucleotides were compared. Different photoreactor configurations were tested for the production of a phosphorylated compound and a flat reactor selected. The presence of inorganic pyrophosphate in the reaction medium was shown to improve the synthesis of ATP 1.4 times. Using the optimal conditions described here, the total G-6P production was 50-fold higher than in batch reactors.

Oscillatory behavior of a simple kinetic model for proteolysis during cell invasion.

Extracellular proteolysis during cell invasion is thought to be tightly organized, both temporally and spatially. This work presents a simple kinetic model that describes the interactions between extracellular matrix (ECM) proteins, proteinases, proteolytic fragments, and integrins. Nonmonotonous behavior arises from enzyme de novo synthesis consecutive to integrin binding to fragments or entire proteins. The model has been simulated using realistic values for kinetic constants and protein concentrations, with fibronectin as the ECM protein. The simulations show damped oscillations of integrin-complex concentrations, indicating alternation of maximal adhesion periods with maximal mobility periods. Comparisons with experimental data from the literature confirm the similarity between this system behavior and cell invasion. The influences on the system of cryptic functions of ECM proteins, proteinase inhibitors, and soluble antiadhesive peptides were examined. The first critical parameter for oscillation is the discrepancy between integrin affinity for intact ECM proteins and the respective proteolytic fragments, thus emphasizing the importance of cryptic functions of ECM proteins in cell invasion. Another critical parameter is the ratio between proteinase and the initial ECM protein concentration. These results suggest new insights into the organization of the ECM degradation during cell invasion.

Influence of light on long-term ADP phosphorylation.

ADP phosphorylation coupled to cyclic electron transport was studied over a long period using immobilized chromatophores from Rhodopseudomonas capsulata. Photophosphorylation of ADP as a function of time was continuously measured under different conditions of continuous illumination and concentration of oxygen. Using a red cutoff filter, it was possible to sustain ADP phosphorylation at a maximal rate for more than 10 days, whereas inactivation had always been observed after 5 days when white light was used. The influence of light nature on inactivation was demonstrated using photopigment absorption spectra.