Covalent Binding of an NAD Analogue to Liver Alcohol Dehydrogenase Resulting in an Enzyme‐Coenzyme Complex not Requiring Exogenous Coenzyme for Activity

Publisher Website
Google Scholar
Abstract
The NAD analog, N6-[N-(6-aminohexyl)carbamoylmethyl]-NAD, was covalently bound to horse liver alcohol dehydrogenase [EC 1.1.1.1] in a carbodiimide-mediated reaction and in such a way that it was active with the very same enzyme molecule to which it was coupled. The degree of substitution, i.e., the number of NAD analoges per enzyme subunit, could be varied (0.3-1.6). In 1 preparation 1.6 coenzyme molecules were bound/subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. Every 4th active site of this preparation was provided with a covalently bound functioning coenzyme analog, and that this analog had a cycling rate of about 40,000 cycles/h in a coupled substrate assay. The presence of the covalently bound coenzyme made the active sites difficult to inhibit with a competitive inhibitor. For example, 10 mM AMP inhibited the activity of the preparation by 50%, whereas a reference system containing native alcohol dehydrogenase was inhibited by 80% in spite of the fact that the reference system contained about 20,000 times as high a concentration of coenzyme.