Open AccessAffinity Laeling of Horse‐Liver Alcohol Dehydrogenase by 3‐Chloroacetylpyridine – Adenine Dinucleotide
Author(s) -
FOUCAUD Bernard,
BIELLMANN JeanFranscois
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb05610.x
Subject(s) - chemistry , alcohol dehydrogenase , cofactor , ternary complex , nad+ kinase , dissociation constant , stereochemistry , enzyme , dehydrogenase , biochemistry , receptor
3‐Chloroacetyl‐pyridine‐adenine dinuleotide (cla 3 PdAD + ) is a reative NAD + analogue with the highest possible conservation of the coenzyme structure on its pyridine moiety; it has been designed to alkylate an enzyme catalytic group from the active site. It interactions with the horse liver alcohol dehydrogenase as well as the interaction of structurally related compounds with this enzyme have been investigated for their specificity and their selectivity. The following results have been obtained. (1) clac 3 PdAD + is active as coenzyme and its isosteric analogue 3‐propionylpyridine‐adenine dinucleotide (pp 3 PdAD + ) does not display cooperative properties in its activity and binding with the horse liver alcohol dehydrogenase, (2) The horse liver alcohol dehydrogenase is irreversily inactivated by clac 3 PdAD + subsequently to the formation of a specific reversible binarycomplex, with a K d of 11 ± 3 μM: NAD+ protects the enzyme in a strictly competitive way, with a K d of 67μM at pH 7.5, 22°C. No inactivation occurs with 3‐chloroacetylpyridine and/with 4‐chloroacetylpyridine‐adenine dinucleotide (clac 4 PdAD + ). (3)The pH dependence of the rate of the enzyme inactivation by clac 3 PdAD + reflects the variation with pH of the dissociation constant of the coenzyme analogue; this variation is similar to that of NAD + . (4) The formation of ternary complexes with ethanol and pyrazole increases the rate of enzyme inactivation, consistently with the enzyme mechanism. From this effect, their dissociation constant could e calcualted and compared to literature data related to other ternary complexes. In contrast, imidazole is shown to decrease the interaction between clac 3 PdAD + and the horse liver alcohol dehydrogenase. (5)The inactivation of the enzyme biphasic at high clac 3 PAD + concentration, and this iphasicity does not result from a heterogeneity of the preparation of the enzyme or coenzyme analogue. Similarities between this biphasicity and a substrate‐induced biphasicity under certain conditions of inactivation of the enzyme are discussed. It is concluded that clac 3 PdAD + is highly specific, and a valuable tool for structural and mechanistic studies of the horse liver alcohol dehydrogenase.