540 Chemie und zugeordnete Wissenschaften
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The spectral properties of binary complexes of NAD-analogues and fragments therefrom with I.DH from pig heart or ADH from liver and yeast have been investigated. The NADH-analogues were modified by replacing adenine through benzimidazole, benzene or dihydronicotinamide. Additionally adenosine diphosphate ribose, dihydronicotinamide and dihydronicotinamide- ribose pyrophosphate-5"-ribose have been studied.
It has been shown by means of difference spectra that complexes between ADH from horse liver and analogues cause spectral changes in the region of aromatic absorption at 280 nm even when adenine is absent in the analogues. Spectral changes in the other enzymes mentioned are probably due to changes of the n-π* absorption of the adenine ring. The spectral changes upon complexing indicate hydrophobic interaction of the adenine with the enzyme protein. Fluorescence spectra vary in the intensity of the energy transfer band as well as in coenzyme emission depending on variation of the coenzym analogue. Changing of complex formation between protein and analogues at different pH-values are investigated. ADH from yeast, especially, shows a pK around 6 which suggests interaction with histidine imidazole.
A new NAD⊕-isomer was prepared, in which the ᴅ-ribose of the adenosine moiety was sub stituted by the enantiomeric ʟ-ribose. As compared to nicotinamide-adenine-dinucleotide (NAD⊕) and NADH the coenzyme isomer (ᴅ,ʟ)-NAD⊕ and its dihydroform (ᴅ,ʟ)-NADH are far less tightly bound to lactate dehydrogenase and alcohol dehydrogenase from horse liver. In the presence of the second substrate (ᴅ,ʟ)-NAD⊕ and (ᴅ,ʟ)-NADH act as hydrogen acceptor and hydrogen donator, respectively, with lactate dehydrogenase and alcohol dehydrogenases from horse liver and yeast. Compared to NAD⊕ and NADH the Michaelis constants are always increased, the catalytic constants (V/Et) were found to be decreased except for the dihydroform reacting with alcohol dehydrogenase from liver.
[ω- (3-Acetylpyridinio) -n-alkyl] adenosine pyrophosphates are coenzyme analogs of NAD⊕. The adenosine pyrophosphate moiety and the 3-acetylpyridine ring of the analogs are connected by n-alkyl chains of different lengths (ethyl -hexyl). The analogs form strong dissociating complexes with lactate dehydrogenase. The complex formation is predominantly achieved by interaction of the ADP moiety with its respective binding domain at the active site.
The redox potentials of the analogs and NAD are of similar magnitude. The coenzyme function of the analogs depends upon the length of the hydrocarbon chain. Lactate dehydrogenase and alcohol dehydrogenases from yeast and horse liver do not catalize hydrogen transfer from their substrates to any other alkyl analog but [4- (3-acetylpyridinio)-n-butyl] adenosine pyrophosphate, aldehyde dehydrogenase from horse liver catalizes hydrogen transfer from acetaldehyde to the pentyl derivative and glyceraldehyde-3-phosphate dehydrogenase catalizes hydrogen transfer to both analogs. In no case, hydrogen transfer from or to one of the 3-acetylpyridine-n-alkyl analogs proceeded with a velocity comparable to NAD or its 3-acetylpyridine analog. The results show that the nicotinamide bound ribose in NAD is involved in the binding and the activation of the coenzyme.