AN ENZYMATICALLY ACTIVE ARTIFICIAL REDOX COENZYME BASED ON A SYNTHETIC DYE TEMPLATE

An analogue of the reactive textile dye, Cibacron Blue F3G-A, has been modified with nicotinamide to produce an artificial redox coenzyme, B lue N-3, whose properties reflect the dual presence of a blue anthraqu inone chromophore and an N-substituted nicotinamide ring. The reduced coenzyme generated concomitantly with the oxidation of alcohols by hor se liver alcohol dehydrogenase (HLADH) displayed difference absorbance (Delta lambda(max) 333 nm) and H-1-NMR spectra consistent with the pr esence of a 1,4-dihydro-nicotinamide ring. The artificial coenzyme med iated the HLADH-catalyzed oxidation of primary aliphatic and cyclic al cohols with pentan-1-ol being the best cosubstrate of those tested and exhibiting a turnover of 8.5% of that shown by NAD(+) at pH 9.0 and 2 5 degrees C. The apparent Michaelis constant (K-m for Blue N-3 (38 mu M) with ethanol as cosubstrate was similar to that of NAD(+) (10 mu M) determined with HLADH and the reaction followed a similar ordered mec hanism. The relative catalytic efficiency [k(cat)/K-m(M(-1)s(-1))] of Blue N-3 was 0.4% of that shown by NAD(+) under these conditions. The artificial coenzyme also displayed activity with human liver beta(1) b eta(1) alcohol dehydrogenase and sheep liver sorbitol dehydrogenase, t wo members of the same long-chain/polyol dehydrogenase family of enzym es. The artificial coenzyme appears to bind to these enzymes, inducing a conformational change and producing a catalytically competent compl ex in a manner similar to the natural coenzyme. (C) 1997 by Elsevier S cience Inc.