OPTICAL SPECTROSCOPY OF NICOTINOPROTEIN ALCOHOL-DEHYDROGENASE FROM AMYCOLATOPSIS-METHANOLICA - A COMPARISON WITH HORSE LIVER ALCOHOL-DEHYDROGENASE AND UDP-GALACTOSE EPIMERASE

The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alco hol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm. Reduced enzyme-bound pyridine dinucleotide could be reversibly oxi dized by acetaldehyde. The fluorescence excitation spectrum for NADH b ound to the enzyme has a maximum at 325 nm. Upon excitation at 290 nm, energy transfer from tryptophan to enzyme-bound NADH was negligible. The fluorescence emission spectrum (excitation at 325 nm) for NADH bou nd to the enzyme has a maximum at 422 nm. The fluorescence intensity i s enhanced by a factor of 3 upon binding of isobutyramide (K-d = 59 mu M). Isobutyramide acts as competitive inhibitor (Ki = 46 mu M) with r espect to the electron acceptor NDMA (N,N-dimethyl-p-nitrosoaniline), which binds to the enzyme containing the reduced cofactor. The nonreac tive substrate analogue trifluoroethanol acts as a competitive inhibit or with respect to the substrate ethanol (K-i = 1.6 mu M), which binds to the enzyme containing the oxidized cofactor. Far-UV circular dichr oism spectra of the enzyme containing NADH and the enzyme containing N AD(+) were identical, indicating that no major conformational changes occur upon oxidation or reduction of the cofactor. Near-UV circular di chroism spectra of NADH bound to the enzyme have a minimum at 323 nm ( Delta epsilon = -8.6 M-1 cm(-1)). The fluorescence anisotropy decay of enzyme-bound NADH showed no rotational freedom of the NADH cofactor. This implies a rigid environment as well as lack of motion of the fluo rophore. The average fluorescence lifetime of NADH bound to the enzyme is 0.29 ns at 20 degrees C and could be resolved into at least three components (in the range 0.13-0.96 ns). Upon binding of isobutyramide to the enzyme-containing NADH, the average excited-state Lifetime incr eased to 1.02 ns and could be resolved into two components (0.37 and 1 .11 ns). The optical spectra of NADH bound to nicotinoprotein alcohol dehydrogenase have blue-shifted maxima compared to other NADH-dehydrog enase complexes, but comparable to that observed for NADH bound to hor se liver alcohol dehydrogenase. The fluorescence lifetime of NADH boun d to the nicotinoprotein is very short compared to enzyme-bound NADH c omplexes, also compared to NADH bound to horse Liver alcohol dehydroge nase. The cofactor-protein interaction in the nicotinoprotein alcohol dehydrogenase active site is more rigid and apolar than that in horse liver alcohol dehydrogenase. The optical properties of NADH bound to n icotinoprotein alcohol dehydrogenase differ considerably from NADH (ti ghtly) bound to UDP-galactose epimerase from Escherichia coli. This in dicates that although bath enzymes have NAD(H) as nonexchangeable cofa ctor, the NADH binding sites are quite different.