Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole.
Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]-NAD+ in order to alkylate the carboxylic acid group of Asp-273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prosthetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated.
Goulas
P
P
Département des Sciences Naturelles, Faculté des Sciences, Pau.
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism; D006736:Horses; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D011720:Pyrazoles / Q000378:metabolism*
D003067:Coenzymes; D011720:Pyrazoles; D009243:NAD; D000426:Alcohol Dehydrogenase