Jean-François Biellmann

Binding of aldose reductase inhibitors : Correlation of crystallographic and mass spectrometric studies

Aldose reductase is a NADP(H)-dependent enzyme, believed to be strongly implicated in the development of degenerative complications of Diabetes Mellitus. The search for specific inhibitors of this enzyme has thus become a major pharmaceutic challenge. In this study, we applied both X-ray crystallography and mass spectrometry to characterize the interactions between aldose reductase and four representative inhibitors: AminoSNM, Imirestat, LCB3071, and IDD384. If crystallography remains obviously the only way to get an extensive description of the contacts between an inhibitor and the enzymatic site, the duration of the crystallographic analysis makes this technique incompatible with high throughput screenings of inhibitors. On the other hand, dissociation experiments monitored by mass spectrometry permitted us to evaluate rapidly the relative gas-phase stabilities of the aldose reductase-inhibitor noncovalent complexes. In our experiments, dissociation in the gas-phase was provoked by increasing the accelerating voltage of the ions (Vc) in the source-analyzer interface region: the Vc value needed to dissociate 50% of the noncovalent complex initially present (Vc50) was taken as a gas-phase stability parameter of the enzyme-inhibitor complex. Interestingly, the Vc50 were found to correlate with the energy of the electrostatic and H-bond interactions involved in the contact aldose reductase/inhibitor (Eel-H), computed from the crystallographic model. This finding may be specially interesting in a context of drug development. Actually, during a drug design optimization phase, the binding of the drug to the target enzyme is often optimized by modifying its interatomic electrostatic and H-bond contacts, because they usually depend on a single atom change on the drug, and are easier to introduce than the hydrophobic interactions. Therefore, the Vc50 may help to monitor the chemical modifications introduced in new inhibitors. X-ray crystallography is clearly needed to get the details of the contacts and to rationalize the design. Nevertheless, once the cycle of chemical modification is engaged, mass spectrometry can be used to select a priori the drug candidates which are worthy of further crystallographic investigation. We thus propose to use the two techniques in a complementary way, to improve the screening of large collections of inhibitors.

Chemical modification of horseradish peroxidase with ethanal-methoxypolyethylene glycol: Solubility in organic solvents, activity, and properties

Abstract The oxidation of polyethylene glycol monomethyl ethers (MW 350, 1990, and 5000) by the Moffatt-Swern method to the corresponding aldehyde is described. These aldehydes are used to modify horseradish peroxidase (HRP) by a reductive amination. The modification of two to three e-NH2 groups of the enzyme was observed. The isoelectric point of the native HRP (pI 8.8) was shifted to pI 5.5 on modification. The modified enzymes have an activity close to that of the native enzyme. Only the enzyme modified with the aldehyde MW 5000 (HRP 5000) was soluble and active in organic solvents like toluene, dioxane, and methylene chloride. In toluene, HRP 5000 was more sensitive to hydrogen peroxide inhibition than in buffer. At room temperature, it is more stable in toluene than in buffer.

Cholesterol oxidase in microemulsion: Enzymatic activity on a substrate of low water solubility and inactivation by hydrogen peroxide

Abstract Cholesterol oxidase from Nocardia erythropolis, Pseudomonas , and Streptomyces species was active in microemulsion in which cholesterol is well solubilized. The activity was stable in nonionic microemulsions whereas in cationic and anionic microemulsions the activity decreased with time. The coupled activity test using horseradish peroxidase which is very stable in microemulsion, was modified. The activity at very low water concentration in nonionic microemulsions increased with the water content. The kinetic constants were determined: the Michaelis constant is in the range 10 to 28 m m in the microemulsions, compared to 10 to 28 μ m in buffer. The maximum velocity was reduced by a factor of 3 to 5 compared to that in buffer. Neither substrate excess nor product inhibition was detected. The preparative oxidation of cholesterol revealed the inactivation of the cholesterol oxidase by hydrogen peroxide. In contrast to glucose oxidase, hydrogen peroxide inactivated cholesterol oxidase in the absence of substrate. Catalase provides protection during the cholesterol oxidation. Microemulsions are very good media in which to perform enzyme catalyzed reactions with substrates of low water solubility. Their use for the reproducible determination of cholesterol should be examined.

Crosslinked crystalline horse liver alcohol dehydrogenase as a redox catalyst: Activity and stability toward organic solvent

Abstract The holoenzyme crystals of horse liver alcohol dehydrogenase-NADH-DMSO complex were crosslinked with glutaraldehyde. A coupled activity test with ethanol and cinnamaldehyde as substrates was performed on the crosslinked enzyme crystals. The enzymatic activity was preserved and the coenzyme was found to be firmly bound to the enzyme crystals. These crystals can be used as redox catalysts with no addition of coenzyme. The crosslinked crystals were more stable toward dimethoxyethane than the enzyme in solution. Zinc ion salts reinforced this stability. Thirty percent of the initial activity was found in a medium containing 84% ( v v ) organic solvents.

RESOLUTION OF ALLYLIC ALCOHOLS BY CHOLESTEROL OXIDASE ISOLATED FROM RHODOCOCCUS ERYTHROPOLIS

Abstract The oxidation of non-steroidal compounds by cholesterol oxidase isolated from Rhodococcus erythropolis is reported for the first time. It was regio-, stereo- and enantio-selective. The enzyme oxidized preferentially the 2-cyclohexenyl-1-alcohols whose configuration is (S) at the reaction center.

Aspartate-β-Semialdehyde Dehydrogenase from Escherichia coli

The substrate binding site of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli was studied by affinity labeling with L-2-amino-4-oxo-5-chloropentanoic acid. The substrate analogue irreversibly inactivates the enzyme with pseudo-first-order kinetics and with a half-of-the-sites reactivity. The substrate aspartate beta-semialdehyde protects the enzyme against the inactivation. A single group is labeled at the active site and is concluded to be the side-chain of a histidine residue. The amino acid sequence around the active site residue was established from a peptic digest of the labeled enzyme: Phe-Val-Gly-Gly-Asp-(modified residue)-Thr-Val-Ser.

Le diptérocarpol—II : Stéréochimie en C-13 et en C-17☆

Abstract Various reactions of ring D of dipterocarpol (hydroxy-dammarenone-II) show this triterpene to have the configuration 13β, 17αH, in accordance with biogenetic speculations.

Preparation of 3-chloroacetylpyridine adenine dinucleotide: An alkylating analogue of NAD+

Analogues of coenzymes with reactive groups have been extensively used to detect the presence of functional amino acid residues in the catalytic site of dehydrogenases. Thus, alkylating analogues of NAD+ have been synthesized: the group at C-3 of nicotinamide of NAD+ has been replaced by a diazonium group [ 11, a diazoacetate [2] or a bromoacetyl group, with an aliphatic chain instead of the ribose [3-61 and the adenine has been replaced by a bromoacetylimidazole [6, 71. As basic residues are invoked in the mechanism of the dehydrogenase for the removal of the proton located at the alcohol during the hydrogen transfer [8,9], it was tempting to attack this basic group from the coenzyme side, by the chloroacetyl group, replacing at C-3 the amide group of the pyridinium ring. Our goal was to synthesize the 3-chloroacetylpyridine adenine dinucleotide analogue of NAD+. This is quite similar to 3-acetylpyridine adenine dinucleotide, which is active as hydrogen acceptor with many dehydrogenases. The similarity of the structure may lead to the activity of this analogue as hydrogen acceptor, as well as to alkylation. This activity is therefore a good indication for the alkylation of a residue in the active site. The preparation of this analogue, and of the 3-propionylpyridine adenine dinucleotide are described here and the preliminary results on the alkylation of some dehydrogenases are briefly reported.

The influence of solvation of the reactions of an allylic carbanion

Aus dem Phenyl-allyl-sulfid (I) erhalt man die Lithium-Verbindung (II), die anschlie= βend mit Methyljodid, Allylbromid und Aceton bei -20°C und bei -78°C umgesetzt wird.

Enzyme and organic solvents: Horse liver alcohol dehydrogenase in non‐ionic microemulsion: Stability and activity

In a microemulsion made with Triton X‐100, the stability of the enzymatic activity was higher than in ionic microemulsions. The stability increased with water content. The kinetic constants (Michaelis constant of NAD+ and maximum velocity) were close to those found in the previously studied microemulsions. The Michaelis constant of NAD+ expressed with respect to the buffer volume was higher than in water. The pH dependence of the kinetic constants in this microemulsion was determined. The activity determined by NAD+ reduction decreased with water content, whereas the redox activity determined via butanol oxidation coupled to retinal reduction was only slightly reduced.