Horse Liver Alcohol Dehydrogenase can Accept NADP⁺ as Coenzyme in High Concentrations of Acetonitrile

Abstract

The coenzyme specificity of horse liver alcohol dehydrogenase assayed in mixtures of acetonitrile and buffer, 0.01 M Tris/HCI 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 acetonitrilehuffer 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.

Redox Enzymes Used in Chiral Syntheses Coupled to Coenzyme Regeneration

“…A racemic mixture of β-hydroxysilanes, 2-trimethyl-1-propanol was optically resolved by using HLADH [226]. Only one of the enantiomers was oxidized and the aldehyde produced was spontaneously degraded to n-propanol (in the presence of HLADH) which provided coenzyme regeneration.…”

Induction of catalytic activity in proteins by lyophilization in the presence of a transition state analogue

“…It can be best illustrated by (bio)molecular imprinting, an approach which enables the introduction of new binding sites into protein molecules (Braco et al, 1990;Dabulis and Klibanov, 1992) and the alteration of an enzyme's catalytic properties such as activity (Mingarro et al, 1995), substrate specificity (Johnsson et al, 1995;O'Rich, and Dordick, 1997) or enantioselectivity (Stahl et al, 1990(Stahl et al, , 1991. However, if ligands can be employed to alter protein conformations in organic solvents in a predictable manner, it should be possible to use this approach for the induction of ''de novo'' catalytic activity in existing proteins via imprinting with transition state analogues (TSA) (Lerner et al, 1991).…”

Influence of water‐miscible organic solvents on kinetics and enantioselectivity of the (R,‐specific alcohol dehydrogenase from Lactobacillus brevis

“…KET4B1 [16], Thermoanaerobacter brockii [17] and Thermoanaerobacter ethanolicus [18]. Furthermore, the addition of the water-miscible co-solvent acetonitrile was used to alter the cofactor specificity of a horse liver ADH (HL-ADH) [19]. Another interesting area for investigation is the interaction of organic solvents on kinetic parameters of enzyme reactions.…”