The interactions of artificial coenzymes with alcohol dehydrogenase and other NAD(P)(H) dependent enzymes |
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Institution: | 1. Oncogenomics and Cancer Proteomics Laboratory, Autonomous University of Mexico City, Mexico;2. Institute of Breast Diseases — FUCAM, Mexico City, Mexico;3. Molecular Biomedicine Program and Biotechnology Network, National School of Medicine and Homeopathy, National Polytechnic Institute, Mexico City, Mexico;4. Translational Medicine Laboratory, National Institute of Cancerology, Mexico City, Mexico;5. Laboratory of Translational Cancer Research and Cellular Therapy, Oncology Hospital, Medical Center Siglo XXI, Mexico City, Mexico;6. Carcinogenesis Laboratory, National Institute of Cancerology, Mexico City, Mexico;7. Lung Cancer Laboratory, National Institute of Respiratory Diseases, Mexico City, Mexico |
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Abstract: | The interactions of CL4, 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)+. CL4, 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°C. HLADH demonstrated a Vmax 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. |
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