The aspartokinase I-homoserine dehydrogenase I of Escherichk coli K 12, composed of six subunits of molecular weight 60,000, binds cooperatively six molecules of L-threonine. A maximum of three molecules of NADP+ or NADPH, measured by a number of techniques, is bound in the presence or in the absence of threonine.Characteristic effects of L-threonine on the protein include a perturbation of its absorption and fluorescence spectra and a quenching of the fluorescence of the protein-NADPH complex. These findings are discussed in view of the structure of the protein.Physical and chemical studies of the protein carrying the aspartokinase I and homoserine dehydrogenase I of E . coli K12 [i, 21 suggest that its six constitutive subunits are identical. The two enzymic functions imply binding sites for their respective substrates, for activating cations (K+ and Mg++) and for the allosteric inhibitor, L-threonine. Kinetic data gave an approximation of the affinities for the different ligands ; threonine and the pyridine nucleotides were found to be most amenable to study. The stoichiometry of their binding is presented here and discussed in relation with the subunit structure. Effects of threonine upon the ultraviolet spectrum of the protein and the fluorescence of its complex with NADPH are also demonstrated.
MATERIAL AND METHODS
Preparation of the ProteinThe pure, electrophoretically homogeneous protein was obtained from extracts of E . wli K12 as described by Truffa-Bachi et al. [l]. The specific activity of homoserine dehydrogenase was a t least 47 units per mg in fresh preparations, and the experiments described here were performed on freshly purified protein unless specified. Enzymic activities were measured and expressed as described in [i].
BuffersBuffer P contained 20 mM potassium phosphate pH 7.2, 2 mM magnesium titriplex and 1 mM dithiothreitol. Buffer A has been described earlier [ l ] and is actually buffer P plus 0.15 M KC1 and 4mM m-threonke. Buffer B is buffer P plus 0.15 M KC1.The protein resuspended from 5001, ammonium sulfate precipitate was equilibrated with buffer by passing it on a small column of Sephadex G 25; exchanging buffers was done the same way.
Chemicals and RadiochemicalsThe salts and chemicals used were the same as in [i]. L-Threonine (allo-free) was obtained from Fluka, NADP+ and NADPH from Sigma.~-[~4C]Threonine (uniformly labelled, 81.6 mC/ m o l e ) was obtained from the Commissariat 21 1'Energie Atomique, [carbonyl-14C]nicotinamide dinucleotide phosphate 18.7 mC/mmole, from the Radiochemical Centre (Amersham, England). The purity of the labelled nucleotides was checked by thin layer chromatography.
Protein DeterminationProtein was determined by biuret related to dry weight by the following method: about 5 mg of purified protein was dialyzed for 2days against distilled water, and lyophilized. Two fractions were then weighed on a microbalance and subjected to the biuret reaction. The remainder was analyzed for C, H, N, content on a Technicon CHN Analyzer. The measured content in carbon...