Dual nucleotide specificity of bovine glutamate dehydrogenase. The role of negative co-operativity.
The thionicotinamide analogues of NAD+ and NADP+ were shown to be good alternative coenzymes for bovine glutamate dehydrogenase, with similar affinity and approx. 40% of the maximum velocity obtained with the natural coenzymes. Both thionicotinamide analogues show non-linear Lineweaver-Burk plots, which with the natural coenzymes have been attributed to negative co-operativity. Since the reduced thionicotinamide analogues have an isosbestic point at 340nm and have an absorption maximum at 400nm, it is possible to monitor reduction of natural coenzyme and thionicotinamide analogue simultaneously by dual-wavelength spectroscopy. When glutamate dehydrogenase is presented with NADP+ and thio-NADP+ simultaneously, the enzyme oligomer senses saturation of its coenzyme-binding sites irrespective of the exact nature of the coenzyme and locks the oligomer into its highly saturated form even when low saturation of the monitored coenzyme is present. These experiments substantiate the suggestion that glutamate dehydrogenase shows negative co-operativity in its catalytically active form.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003641:Deamination; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D013053:Spectrophotometry; D013873:Thionucleotides / Q000378:metabolism
D005971:Glutamates; D013873:Thionucleotides; D009243:NAD; C015541:thionicotinamide adenine dinucleotide phosphate; C003451:thionicotinamide adenine dinucleotide; D009249:NADP; D005969:Glutamate Dehydrogenase