Study of coenzyme binding site of octopine dehydrogenase using analogues of NAD+.
The binding of the coenzyme to octopine dehydrogenase was investigated by kinetic and spectroscopic studies using different analogues of NAD+. The analogues employed were fragments of the coenzyme molecule and dinucleotides modified on the purine or the pyridine ring. The binding of ADPribose is sufficient to induce local conformational changes necessary for the good positioning of substrates. AMP, ADP, NMN+ and NMNH do not show this effect. Analogues modified on the purine ring such as nicotinamide deaminoadenine dinucleotide, nicotinamide--8-bromoadenine dinucleotide, nicotinamide--8-thioadenine dinucleotide and nicotinamide 1: N6-ethenoadenine dinucleotide bind to the enzyme and give catalytically active ternary complexes. Modifications of the pyridine ring show an important effect on the binding of the coenzyme as well as on the formation of ternary complexes. Thus, the carboxamide group can well be replaced by an acetyl group and also, though less efficiently, by a formyl or cyano group. However more bulky substituents such as thio, chloroacetyl or propionyl groups prevent the binding. The analogues bearing a methyl group in the 4 or 5 position, which are competitive inhibitors, are able to give binary by not ternary complexes. The case of 1,4,5,6-tetrahydronicotinamide--adenine dinucleotide which does not give ternary complexes like NADH is discussed. The above findings show that the pyridine and adenine parts are both involved in the binding of the coenzyme and of the substrate to octopine dehydrogenase. The nicotinamide binding site of this enzyme seems to be the most specific and restricted one among the dehydrogenases so far described. The protective effects of coenzyme analogues towards essential -SH group were also studied.
D000594:Amino Acid Oxidoreductases; D000600:Amino Acids, Dicarboxylic; D000818:Animals; D001665:Binding Sites; D005399:Fishes; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000031:analogs & derivatives* / Q000494:pharmacology; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D000600:Amino Acids, Dicarboxylic; D008024:Ligands; D009243:NAD; D000594:Amino Acid Oxidoreductases; D000587:Oxidoreductases Acting on CH-NH Group Donors; C021903:D-octopine dehydrogenase