Abstract
The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.
Highlights
In plants and bacteria, the first and the third steps of methionine and threonine biosynthesis are catalyzed by isoforms of the bifunctional enzyme, aspartate kinase (AK)1 (EC 2.7.2.4)-homoserine dehydrogenase (HSDH) (EC 1.1.1.3) [1, 2]
Regulatory Domain of AK-HSDH—It was previously shown by internal sequence comparison that the E. coli threoninesensitive AK-HSDH regulatory domain is composed of two homologous subdomains [9]
This feature is found for A. thaliana threonine-sensitive AK-HSDH (Fig. 1)
Summary
The assay mixture contained 100 mM Tris-HCl, pH 8.0, 400 mM hydroxylamine-KOH, pH 8.0, 0 –50 mM aspartate-KOH, pH 8.0, 0 – 40 mM ATP-KOH, pH 8.0, 20 mM MgCl2, This paper is available on line at http://www.jbc.org. Allosteric Control of Aspartate Kinase-Homoserine Dehydrogenase and 150 mM KCl. The reaction was initiated by the addition of AKHSDH enzyme (final volume, 1 ml) and was carried out at 37 °C for 10 min. Enzyme activity was expressed as micromoles of NADPH produced per minϪ1 per mgϪ1 of protein using an ⑀340 nm of 6250 MϪ11⁄7cmϪ1. In Vitro Assays of HSDH in the Forward Direction—Enzyme activity was expressed as micromoles of NADPH transformed per minϪ1 per mgϪ1 of protein using an ⑀340 nm of 6250 MϪ11⁄7cmϪ1. A search of homologies was carried out with the Pfam protein data base [25]
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