PH-dependent intermediate plateaux in the kinetics of the reaction catalyzed by “biosynth” l-threonine dehydratase of Escherichia coli K-12

Abstract

1. 1.|A method for the determination of l-threonine dehydratase ( l-threonine hydro-lyase (deaminating), EC 4.2.1.16) activity, based on measurements of the amount of 2-oxobutyrate formed in the threonine dehydratase reaction by means of an auxiliary lactate dehydrogenase system (EC 1.1.1.27), was described. 2. 2.|The kinetics of the reaction catalyzed by the “biosynthetic” l-threonine dehydratase in a crude extract from Escherichia coli K-12 cells were studied. Four inflexion points ( i.e., two intermediate plateaux) on the plot of initial reaction rate (ν) versus initial substrate concentration ([ S] 0) were distinctly revealed at pH > 9. 3. 3.|The allosteric inhibitor l-isoleucine, at certain pH values, displaced the sigmoid shape of the plot of v versus [ S] 0 to higher substrate concentrations; the magnitude of the maximal reaction rate ( V) did not change. At high concentrations of enzyme protein, l-isoleucine, at low concentrations, removed the intermediate plateau on the plot of v versus [ S] 0 and hence “activated” the enzyme in the region of high substrate concentrations, whilst the second intermediate plateau was demonstrated in these curves as in the usual sigmoidal plots. 4. 4.| dl-Norvaline “normalized” the kinetics of the reaction catalyzed by the enzyme and at the same time decreased the value of V. 5. 5.|Treatment of the enzyme with low concentrations of HgCl 2 (7.5·10 −5 M) led to the complete desensitization of the enzyme in relation to the inhibitory effect of l-isoleucine at pH 9.6 and “normalized” the effects of dl-norvaline on the reaction kinetics. The plot of v versus [ S] 0 for the desensitized enzyme was characterized by the absence of the inflexion points (intermediate plateaux) which were characteristic for the native enzyme at this pH value, although the plot did not become hyperbolic. Treatment of the enzyme at pH 8.2 with lower concentrations of HgCl 2 (1.0·10 −5 M) led to selective desensitization, namely, the sensitivity to the “normalizing” effect of dl-norvaline was completely lost but the sensitivity to the inhibitory effect of l-isoleucine was partially restored. It was suggested that the enzyme molecule possesses two spatially divided binding sites for these allosteric effectors. 6. 6.|The shape of the plot of v versus [ S] 0 depends on the concentration of enzyme protein, namely, at low enzyme concentrations two intermediate plateaux were revealed on the plot of v versus [ S] 0. At the same time, an increase in enzyme concentration led to the annihilation of the intermediate plateau demonstrated at low substrate concentrations and to the displacement of the second intermediate plateau found at high substrate concentrations towards lower substrate concentrations. 7. 7.|Low concentrations of l-isoleucine removed this second intermediate plateau only at high enzyme concentrations, and hence “activated” the enzyme at about the substrate concentrations at which the second intermediate plateau was revealed. 8. 8.|The data obtained were discussed, taking into consideration the possible effects of allosteric ligands on the dissociation of the enzyme oligomer and on the conformation states of subunits in the oligomer itself.