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Limited proteolysis of aspartokinase I-homoserine dehydrogenase I from Escherichia coli by type VI protease from Streptomyces griseus yields five proteolytic fragments, three of which are dimeric, the other two being monomeric. One of the monomeric fragments (27 kilodaltons) exhibits residual aspartokinase activity, while the second one (33 kilodaltons) possesses residual homoserine dehydrogenase activity. The smallest of the dimeric species (2 X 25 kilodaltons) is inactive; the two other dimers exhibit either only homoserine dehydrogenase activity (2 X 59 kilodaltons) or both activities (hybrid fragment, 89 + 59 kilodaltons). This characterization of the proteolytic species in terms of molecular weight, subunit structure, and activity leads to the proposal of a triglobular model for the native enzyme. In addition, the time course of the formation of the various fragments was followed by measuring enzymatic activity and performing gel electrophoretic analysis of the protein mixture at defined time intervals during proteolysis. On the basis of the results of these studies, a reaction scheme describing the succession of events during proteolysis is given.

Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) utilize pretyrosine [fl-(1-carboxy-4-hydroxy-2,5cyclohexadien-1-yl) alanine] as an intermediate in L-tyrosine biosynthesis. Pretyrosine is fonned from prephenate via the activity of at least one species of aromatic aminotransferase which is significantly greater with prephenate as substrate than with either phenylpyruvate or 4-hydroxyphenylpyruvate. Pretyrosine dehydrogenase, capable of converting pretyrosine to L-tyrosine, has been partially purified from all three species. Each of the three pretyrosine dehydrogenases is catalytically active with either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as cofactors. The Km values for nicotinamide adenine dinucleotide phosphate in C. glutamicum and B. flavum are 55 AM and 14.2 ,uM, respectively, and corresponding Km values for nicotinamide adenine dinucleotide are 350,M and 625,uM, respectively. The molecular weights of pretyrosine dehydrogenase in C. glutamicum and in B. flavum are both about 158, 000, compared with 68,000 molecular weight in B. ammoniagenes. In all three species the enzyme is not feedback inhibited by L-tyrosine. Results obtained with various auxotrophic mutants, which were used to manipulate intemal concentrations of L-tyrosine, suggest that pretyrosine dehydrogenase is expressed constitutively. Pretyrosine dehydrogenase is quite sensitive to p-hydroxymercuribenzoic acid, complete inhibition being achieved at 10 to 25 uM concentrations. This inhibition is readily reversed by thiol reagents such as 2mercaptoethanol. Coryneform organisms, like species of blue-green bacteria,

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Wild-type Brevibacterium flavum has shown to possess arogenate dehydrogenase activity and I prephenate dehydrogenase, thereby providing presur evidence that arogenate (previously named "pretyrosine' obligatory intermediate of L-tyrosine biosynthesis. A s enzymological pattern has been discerned in extracts mad wild-type cultures of various species of cyanobacteria. cation of rigorous molecular genetic criteria in confirm of the exclusive role of arogenate in L-tyrosine synthes made possible by the isolation of an auxotrophic muti hibiting a nutritional requirement for L-tyrosine. The n was found to lack activity for arogenate dehydrogenase accumulate substantial amounts of arogenate behind the I block during starvation for L-tyrosine. arogenate branchlet for L-tyrosine biosynthesis. Wild-type cells of Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes lack prephenate dehydrogenase activity and were shown to contain prephenate aminotransferase and arogenate dehydrogenase, the two enzyme activities illustrated above.More rigorous establishment of conclusions based upon data derived from wild-type cells would be provided by the demonstration that tyrosine auxotrophy corresponds to the mutational loss of arogenate dehydrogenase and that arogenate accumulates behind the mutant block. The latter approach has been fulfilled in B. flavum, and this system now offers the most rigorous documentation to date of an exclusive role in vivo of the arogenate branchlet for L-tyrosine biosynthesis. MATERIALS AND METHODS

Species ofcoryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) are capable of transaminating all three of the aromatic pathway intermediates: prephenate, phenylpyruvate, and 4-hydroxyphenylpyruvate. Two molecular species of aromatic aminotransferase (denoted aminotransferase I and aminotransferase II) were partially purified from C.

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

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