Glutamate déshydrogénase.

The pig liver glutamic dehydrogenase has been studied by difference spectroscopy and shows two diphosphopyridine nucleotide binding sites per protomer of molecular weight 52 000. A first NADH molecule binds on an adenylic site (Ka = 4.2 mM-1), and a second molecule on an nico- tinamide site (Kn = 13 mM-1). NADPH binds only on the nicotinamide site (K = 21 mM-1). The adenylic site does not only strongly bind ADP (K = 2800 mM-1), but also ADP-ribose, ATP, AMP, NADH, NAD, ApA, GpA, with a decreasing affinity; ApG and GTP does brad on tiffs site which is specific of the adenosine 5'-phosphate group. The high ionic strength decreases ADP binding. The linkage between the nicotinamide site and NADH induces a positive Cotton effect in the range of the absorption band of the reduced nicotinamide group; moreover the absorption spectra of bound NADH is shifted about 5 nm to tho greater wavelengths with an hypochromism of 6%, with respect to free NADH. The evidence for two distinct binding sites is given by: (a) the existence of two difference spectra: the first due to the nicotinamide chromophore perturbation and the second due to the adenylic chromophore perturbation. This last is sitar to that obtained with ADP, AMP, ATP; etc.; (b) the competition between ADP and NADH only on the adenylic site; (c) the fact that the saturation curves obtained from measurements made in the absorption range of adenosine and reduced nicotinamide chromophores are different and square with the theoretical saturation curves of two distinct sites per protomer. NAD also binds on the adenylic site in competition witch ADP, whereas NADP and NADPH do not induce any perturbation of the adenosine chromophore. All these results are in good agreement with the interpretations given by Frieden to explain the effects observed in the steady-state enzymatic kinetics at high concentrations of coenzyme. [ABSTRACT FROM AUTHOR]