Reversible dissociation of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12. The active species is the tetramer

Abstract

Dimers of aspartokinase I/homoserine dehydrogenase 1 from Escherichiu coli K 12 have been isolated under very mild conditions. The dimers which cannot be distinguished from the tetramers by their kinetic properties, reassociate in the presence of potassium ions or L-aspartate. The selective sensitivity of aspartokinase I/homoserine dehydrogenase I to mild proteolytic digestion of dimers has been used to probe the reassociation reaction under the conditions of aspartokinase assay. We demonstrate that rapid reassociation occurs and that the protein species present in the assay when dimers are used to test the activity is tetrameric. These results confirm the previously proposed model for the subunit association of aspartokinase I/homoserine dehydrogenase I.Aspartokinase I/homoserine dehydrogenase I (AK I/ HDH I) is a bifunctional tetrameric protein which catalyses two non-consecutive reactions in the biosynthetic pathway leading from aspartate to threonine and isoleucine [l -31. Each of the four identical polypeptide chains [4, 51 carries the two catalytic activities which are both subject to feedback inhibition by L-threonine [1, 61.Most enzymatic and spectroscopic properties of AK I/ HDH I can be interpreted in terms of a two-state equilibrium between an active and an inactive conformation respectively by different ligands: L-aspartate and K f ions favor the active conformation while L-threonine stabilizes the inactive state of the protein [7, 81. Like many other polymeric enzymes, AK I/HDH I can be found in more than one state of association, and, under certain conditions, the tetramer dissociates into dimers [5]. A number of results indicate that the interprotomeric association involve two types of contacts between subunits and that dissociation of the tetramer into dimers is more easily achieved than dissociation of dimers into monomers [9]. Indeed, dimers retaining at least part of the initial enzymatic activities have been obtained by incubation at low temperature and moderately alkaline pH [5] or in the absence of aspartate and potassium ions [lo, 111. The dissociation is usually accompanied by a loss in sensitivity towards threonine inhibition [12], a result also obtained when the native protein is reacted under mild conditions with sulfhydryl reagents [13]. Dissociation to the monomer is usually accompanied with a loss of enzymatic activity [14].On the other hand, limited proteolysis of native AK I/ HDI I under very mild conditions has yielded a variety of proteolytic fragments with principally a homodimeric HDHCorrespondenc.e to

Purification and Characterization of Lysine-Sensitive Aspartate Kinase from Maize Cell Cultures

“…Carrot AK was also shown to dissociate into independently migrating active species of 100,000 and 150,000 during sucrose density gradient centrifugation (21). Escherichia coli aspartokinase I/homoserine dehydrogenase I also exists in different active aggregation states, a 320,000 tetramer and a 122,000 dimer (25,26 The two subunits resolved from AK Late may be products of two genes or result from in vitro or in vivo modification of a single gene product. At least two genes encode different barley AK isozymes (22); however, barley AK isozymes have not been characterized at the structural level to determine subunit composition.…”

“…The gel filtration column was eluted at 20 mL/h overnight. Fractions containing AK activity were pooled and diluted with one volume of AE1 buffer (25 [v/ v] glycerol, 3 mm DTT). The enzyme was loaded at this higher pH onto the Mono Q HR5/5 column and eluted with a 30-mL KCI gradient (0-500 mM) into 0.5 mL fractions.…”

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase

“…The biochemistry of AKs in bacteria and plants is quite complex. The AKs from Escherichia coli include the bifunctional AK-HDH I which assembles into a 360 kDa tetrameric complex (Veron et al, 1985), while the second bifunctional enzyme (AK-HDH II) is found as a dimer in solution (Dautry-Varsat et al, 1977). The monofunctional AK III from E. coli assembles into a smaller dimer of only 96 kDa (Richaud et al, 1973).…”

Oligomerization and the Sensitivity of Phosphoenolpyruvate Carboxylase to Inactivation by Proteinases

“…The recent report that aspartokinase 1/homoserine dehydrogenase I in the dimeric form was subject to inactivation by pronase type VI, while conversion to the tetramer rendered it much more resistant to cleavage of the protein molecule (13) suggests other mechanisms by which enzymes may be regulated, ' Supported in part by grant DMB 85-15181 from the National Science Foundation.…”