Electron transfer from NADH bound to horse liver alcohol dehydrogenase (NAD+ dependent dehydrogenase): visualisation of the activity in the enzyme crystals and adsorption of formazan derivatives by these crystals.
The crystals of holoenzyme from native and cross-linked alcohol dehydrogenase exhibit electron transfer from NADH to phenazinium methosulfate (PMS), and then to the tetrazolium salt sodium 3,3'-{1-[(phenylamino)carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonate (XXT). The slow dissociation of the cofactor and/or the conformational change associated can now be bypassed. The reduction product, formazan, did not diffuse out of the crystals in buffer and the crystals turned colored. In the presence of dimethyl sulfoxide or dimethoxyethane, the formazan diffused out to the solution. The reaction rates were found to be, respectively, 18% and 15% of the redox reaction rate of ethanol with cinnamaldehyde, close to the activity determined for the enzyme in solution in the presence of dimethoxyethane. The use of system PMS-tetrazolium salt is a useful tool to visualize the activity of dehydrogenases and other electron transferring systems in the crystalline state. The adsorption of formazan by the alcohol dehydrogenase crystals occurs in solution.
Pacaud-Mercier
Karine
K
UMR 7123 Faculte de Chimie, Universite Louis Pasteur, 67000 Strasbourg, France.
Blaghen
Mohamed
M
Lee
Kang Min
KM
Tritsch
Denis
D
Biellmann
Jean-François
JF
D000171:Acrolein / Q000031:analogs & derivatives / Q000737:chemistry; D000327:Adsorption; D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D000818:Animals; D002384:Catalysis; D003460:Crystallization; D004058:Diffusion; D004579:Electron Transport; D005562:Formazans / Q000737:chemistry; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008773:Methylphenazonium Methosulfate / Q000031:analogs & derivatives / Q000737:chemistry; D015394:Molecular Structure; D009243:NAD / Q000737:chemistry*; D010084:Oxidation-Reduction; D013778:Tetrazolium Salts / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry
D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; D009243:NAD; D008773:Methylphenazonium Methosulfate; D000171:Acrolein; D000426:Alcohol Dehydrogenase; C022616:thiazolyl blue; C012843:cinnamaldehyde