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 nicotinamide functionality coupled via a triazine ring to a dibenzenesulphonic acid unit, and of a series of analogues, with HLADH and other dehydrogenases have been compared to those of the natural coenzymes NAD(P)(+). CIA, together with one analogue with one of the sulphonic acid groups shifted by one position and another analogue with a single benzenedisulphonic acid unit, have been shown to be functional mimics of NAD(+) in the oxidation of butan-1-ol by horse Liver alcohol dehydrogenase (HLADH). A combination of discontinuous HPLC-based assays and continuous fluorescence based assays were used to deduce approximate kinetic constants for this reaction, using the artificial coenzymes, at pH 7.5 and 37 degrees C. HLADH demonstrated a V-max with 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 mesenteroides glucose-6-phosphate dehydrogenase and sheep liver sorbitol dehydrogenase; all displayed activity using a range of the biomimetic coenzymes. (C) 1999 Elsevier Science B.V. All rights reserved.