Étude cinétique de changements conformationnels de la l ‐glutamate déshydrogénase provoqués par le couple effecteur GTP + NADH

It is shown by rapid kinetics that when the coenzyme NADH and the effector GTP are added to the enzyme, l ‐glutamate dehydrogenase, several changes occur following a multiphasic process. These changes are observed by a variation in the absorbance, and fluorescence intensity of the fixed nicotinamide ring of NADH, as well as by a variation in the fluorescence intensity of the fluorescent probe, ANS, which is fixed to the protein. By all three methods, two phases are generally observed, the slower phase ( k 2 = 1.5 sec –1 ) being characterized by a change in the molar absorbancy coefficient of the nicotinamide ring (∼15% at 365 nm). The more rapid phase ( k 1 = 60 sec –1 ) is characterized by a decrease in turbidity without any spectral modifications of the fixed NADH. The binding rates of the two ligands NADH and GTP, presented elsewhere, are too rapid to be taken into consideration as being responsible for the steps observed here. The step ( k 1 = 60 sec –1 ) which shows a decrease in turbidity is thus associated with a depolymerization of the polymer (composed of an indefinite number of units of molecular weight 310000). Although only partial, such a dissociation, already known to exist by simple dilution of the enzyme, proceeds with a slower rate ( k = 12 sec –1 ) than that of the dissociation provoked by NADH and GTP. A study was also carried out using the coenzyme NADPH instead of NADH. The amplitude of the phase identified with the depolymerization of the enzyme is similar to that observed for NADH. A striking difference is found in the fact that there is but a negligible modification of the nicotinamide ring of NADPH. It should also be noted the order of incubation of these ligands affects only the rate of depolymerization; in particular, pre‐incubation of the enzyme with NADH, but not with NADPH, notably slows down the rate of depolymerization. The results obtained with NADH and NADPH give support to the hypothesis proposed by Frieden from kinetic studies, that NADH, not NADPH, can bind to two distinct sites, an active site and an effector or regulatory site. Correlation of our results with known kinetic data has enabled us to give a functional aspect to each site. The active site is associated with the depolymerization effect, while the regulatory site is the site which we consider to be responsible for the “protector” and “transconforming” effects. A summary of the observed rates is presented in schematic form, in an effort to understand the mechanism of action of NADH and GTP. The process which culminates in the inhibition of the enzyme activity occurs before the two structural modifications which are measured here, since the inhibitory effect of GTP on the oxidation of the coenzyme appears in less than 2 msec. Preliminary results show that the activator ADP reverses both the depolymerization and the spectral modifications of NADH produced by NADH and GTP.