Preparation and partial characterization of a soluble site-to-site directed enzyme complex composed of alcohol dehydrogenase and lactate dehydrogenase |
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Authors: | Nils Siegbahn Mats-Olle Maånsson Klaus Mosbach |
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Institution: | (1) Pure and Applied Biochemistry, Chemical Center, University of Lund, PO Box 124, S-221 00 Lund, Sweden |
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Abstract: | A soluble, bifunctional enzyme complex has been prepared by crosslinking lactate dehydrogenase and alcohol dehydrogenase with
glutaraldehyde. The crosslinking was performed on a solid phase while the active sites of alcohol dehydrogenase and lactate
dehydrogenase were held adjacent to one another with the aid of a bis-NAD analog. Subsequently, the enzyme complex was released
from the solid phase. The soluble enzyme complex was then purified by using NAD-Sepharose as an affinity adsorbent. Based
on gel filtration experiments, the complex was estimated to consist of one of each dehydrogenase.
By using a third enzyme, lipoamide dehydrogenase, which competes with lactate dehydrogenase for NADH produced by alcohol dehydrogenase,
the effect of site-to-site orientation was studied. It was found that about 83% of the NADH produced by alcohol dehydrogenase
was oxidized by site-to-site oriented lactate dehydrogenase compared to a figure of only about 61% obtained in an identical
system of separate enzymes. This indicates that given two alternative routes, the preference for the one to lactate dehydrogenase
over the one to lipoamide dehydrogenase is enhanced when lactate dehydrogenase and alcohol dehydrogenase are site-to-site
oriented. |
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Keywords: | Reversible immobilization of an enzyme complex oriented enzyme complex reversible immobilization of alcohol dehydrogenase reversible immobilization of the lactate dehydrogenase complex of lactate dehydrogenase reversible immobilization of the alcohol dehydrogenase complex of substrate channeling of an immobilized enzyme complex Bis-NAD and reversibly immobilized enzyme complexes enzyme complex reversible immobilization of complex reversible immobilization of an enzyme dehydrogenases reversible immobilization of complexes of immobilized enzyme complex reversible |
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