Site-to-site directed immobilization of enzymes with bis-NAD analogues.

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Abstract
Lactate dehydrogenase (bovine heart EC 1.1.1.27) and alcohol dehydrogenase (horse liver, EC 1.1.1.1) were crosslinked with glutaraldehyde on agarose beads. The crosslinking was performed while the 2 enzymes were spatially arranged with their active sites facing one another with the aid of a bis-NAD analog. Subsequently the bis-NAD analog was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (pig heart, EC 1.6.4.3), which was also coupled to the same beads and which competes with lactate dehydrogenase for the NADH produced by alcohol dehydrogenase, the effect of site-to-site directed immobilization was studied. Much more NADH than was theoretically expected (50% instead of 19% of produced NADH) was oxidized by lactate dehydrogenase, which indicates that the NADH was preferentially channeled to lactate dehydrogenase due to the juxtapositioned active sites of the 2 enzymes.