Horse liver alcohol dehydrogenase can accept NADP+ as coenzyme in high concentrations of acetonitrile.
The coenzyme specificity of horse liver alcohol dehydrogenase assayed in mixtures of acetonitrile and buffer, 0.01 M Tris/HCl pH 7.4, was investigated. The enzyme could accept NADP+ as coenzyme if it was first bio-imprinted with NADP+, i.e. precipitated from an aqueous buffer with 1-propanol in the presence of NADP+ dried, and then assayed in a system with less than 10% buffer. When assayed in a system with 25% buffer, no activity with NADP+ as coenzyme was observed. The activity was measured with a coenzyme regeneration assay with cinnamoyl alcohol and octylaldehyde as substrates. Other methods to prepare a binary complex of horse liver alcohol dehydrogenase and NADP+, i.e. if the enzyme was immobilized on silica and NADP+ added afterwards or if it was deposited on Celite together with NADP+, failed to show any bio-imprinting effect. The activity of horse liver alcohol dehydrogenase bioimprinted with NADP+ and assayed in acetonitrile/buffer mixtures with less than 10% buffer showed the same or higher activity than an enzyme preparation prepared by bio-imprinting with NAD+. The hypothesis is that the conformation of the active site of horse liver alcohol dehydrogenase is modified to one that is complementary to the ligand present during precipitation and drying. This is possible because of the restricted motility of the enzyme conformation in high concentrations of organic solvent. In the presence of more than 10% buffer the mobility increases in such a way that the imposed conformation with activity towards NADP+ disappears.
Johansson
A
A
Chemical Center, Lund University, Sweden.
Mosbach
K
K
Månsson
M O
MO
D000097:Acetonitriles / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D006736:Horses; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*; D013379:Substrate Specificity
D000097:Acetonitriles; D009249:NADP; D000426:Alcohol Dehydrogenase; C032159:acetonitrile