Biomedical subjects

Yvain Nicolet

Publications and source records attributed to Yvain Nicolet.

4 recordsLinked to original sources

PubMed2003

Photoreversible inhibition of cholinesterases: catalytic serine-labeled caged butyrylcholinesterase.

The photoregulation of the catalytic activity of butyrylcholinesterase (BChE) was investigated by treating the enzyme with a newly developed carbamylating reagent, N-methyl-N-(2-nitrophenyl)carbamoyl chloride, which has proved to be an efficient photoremovable alcohol-protecting group. BChE was efficiently inhibited in a time- and concentration-dependent manner, and the enzyme could be protected against inhibition by active-site-specific ligands (that is, tacrine). The inactivation kinetics showed a biphasic character, which can be analyzed as the result of the existence of two conformational states in solution. Pseudo-irreversible inactivation of BChE, which results from catalytic serine carbamylation, was suggested by recovery of the enzyme activity after dilution and was demonstrated by X-ray crystallography. Remarkably, the 3D structure of the carbamylated BChE conjugate showed a nonambiguous carbamylation of the catalytic serine residue as the only chemical modification on the protein. The photoreversibility of the enzyme inactivation was analyzed by irradiating the inactivated enzyme at 365 nm and was shown to reach completion in some conditions. The efficient and specific "caging" of BChE, together with the availability of carbamylated BChE crystals, will offer a unique possibility to study the catalytic properties of this enzyme by kinetic crystallography after cryophotolytic uncaging of the enzyme conjugate crystals.

Animals↗

PubMed2003

Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products.

Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser(198) has been modeled as bound butyrate.

Acetylcholinesterase↗

PubMed2002

Fe-only hydrogenases: structure, function and evolution.

Hydrogenases are enzymes capable of catalyzing the oxidation of molecular hydrogen or its production from protons and electrons according to the reversible reaction: H(2)<==>2H(+)+2e(-). Most of these enzymes fall into to major classes: NiFe and Fe-only hydrogenases. Extensive spectroscopic, electrochemical and structural studies have shed appreciable light on the catalytic mechanism of hydrogenases. Although evolutionarily unrelated, NiFe and Fe-hydrogenases share a common, unusual feature: an active site low-spin Fe center with CO and CN coordination. We have recently focused our attention on Fe-hydrogenases because from structural studies by us and others, it appears to be a simpler system than the NiFe counterpart. Thus the primary hydrogen binding site has been identified and plausible, electron, proton and hydrogen pathways from and to the buried active site may be proposed from the structural data. The extensive genome sequencing effort currently under way has shown that eukaryotic organisms contain putatively gene coding sequences that display significant homology to Fe-hydrogenases. Here, we summarize the available evidence concerning the mechanism of these enzymes and carry out a structural comparison between Fe-hydrogenases and related proteins of unknown metal content from yeast, plant, worm, insect and mammals.

Amino Acid Sequence↗

PubMed2002

Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization.

Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 A resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 A, c = 124.9 A, giving a Vm of 2.73 A3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general.

Amino Acid Sequence↗