The interactions of artificial coenzymes with alcohol dehydrogenase and other NAD(P)(H) dependent enzymes

The interactions of CLA, a biomimetic analogue of NAD(+) comprising a nicot inamide functionality coupled via a triazine ring to a dibenzenesulphonic a cid unit, and of a series of analogues, with HLADH and other dehydrogenases have been compared to those of the natural coenzymes NAD(P)(+). CIA, toget her with one analogue with one of the sulphonic acid groups shifted by one position and another analogue with a single benzenedisulphonic acid unit, h ave been shown to be functional mimics of NAD(+) in the oxidation of butan- 1-ol by horse Liver alcohol dehydrogenase (HLADH). A combination of discont inuous HPLC-based assays and continuous fluorescence based assays were used to deduce approximate kinetic constants for this reaction, using the artif icial coenzymes, at pH 7.5 and 37 degrees C. HLADH demonstrated a V-max wit h the most active analogue which was 4% of that with NAD(+). The substrate specificity of HLADH using these coenzymes was found to change relative to that using the natural coenzyme. Activity was sought from a range of other dehydrogenases: Bacillus megaterium glucose dehydrogenase, Leuconostoc mese nteroides glucose-6-phosphate dehydrogenase and sheep liver sorbitol dehydr ogenase; all displayed activity using a range of the biomimetic coenzymes. (C) 1999 Elsevier Science B.V. All rights reserved.