Lactate dehydrogenase (L-lactate:NAD' oxidoreductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol: NAD' oxidoreductase, EC 1.1.1.1) have been crosslinked with glutaraldehyde on agarose beads. The crosslinking was performed while the two enzymes were spatially arranged with their active sites facing one another with the aid of a bis-NAD analogue. Subsequently the bis-NAD analogue was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, 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-tosite directed immobilization was studied. It was found that 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 two enzymes.Several coimmobilized multistep enzyme systems have been described in the literature (1-6). For example, an immobilized system composed of the sequence malate dehydrogenase/citrate synthase showed a consistantly higher overall steady-state rate (6). More recently, a four-enzyme sequence (7) and even the enzymes of a complete metabolic cycle, the urea cycle, have been coimmobilized to supports (8). In the latter case, the immobilized cyclic enzyme system again was more efficient than the corresponding soluble system. These effects have been partly attributed to the close proximity of the enzymes and partly to the diffusional restrictions imposed by the Nernst unstirred layer around the enzymes (2, 9).
Lactate dehydrogenase (L-lactate:NAD' oxidoreductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol: NAD' oxidoreductase, EC 1.1.1.1) have been crosslinked with glutaraldehyde on agarose beads. The crosslinking was performed while the two enzymes were spatially arranged with their active sites facing one another with the aid of a bis-NAD analogue. Subsequently the bis-NAD analogue was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, 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-tosite directed immobilization was studied. It was found that 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 two enzymes.Several coimmobilized multistep enzyme systems have been described in the literature (1-6). For example, an immobilized system composed of the sequence malate dehydrogenase/citrate synthase showed a consistantly higher overall steady-state rate (6). More recently, a four-enzyme sequence (7) and even the enzymes of a complete metabolic cycle, the urea cycle, have been coimmobilized to supports (8). In the latter case, the immobilized cyclic enzyme system again was more efficient than the corresponding soluble system. These effects have been partly attributed to the close proximity of the enzymes and partly to the diffusional restrictions imposed by the Nernst unstirred layer around the enzymes (2, 9).
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