Open AccessCovalent Binding of an NAD Analogue to Liver Alcohol Dehydrogenase Resulting in an Enzyme‐Coenzyme Complex not Requiring Exogenous Coenzyme for Activity
Author(s) -
MÅNSSON MatsOlle,
LARSSON PerOlof,
MOSBACH Klaus
Publication year - 1978
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1978.tb12328.x
Subject(s) - cofactor , nad+ kinase , alcohol dehydrogenase , chemistry , enzyme , covalent bond , biochemistry , coenzyme a , stereochemistry , dehydrogenase , glycerol 3 phosphate dehydrogenase , organic chemistry , reductase
1 The NAD analogue, N 6 ‐[ N ‐(6‐aminohexyl)carbamoylmethyl]‐NAD, was covalently bound to horse liver alcohol dehydrogenase 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. 2 The degree of substitution, i.e. the number of NAD analogues per enzyme subunit, could be varied (0.3–1.6). In one preparation 1.6 coenzyme molecules were bound per subunit; the alcohol dehydrogenase activity of this preparation was 40% of the activity obtained after addition of free NAD in excess. 3 It was calculated that every fourth active site of this preparation was provided with a covalently bound functioning coenzyme analogue, and that this analogue had a cycling rate of about 40000 cycles/h in a coupled substrate assay. 4 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 20000 times as high a concentration of coenzyme.