Dehydrogenase Fragment Research Articles

The primary structure of Escherichia coli K12 aspartokinase I-homoserine dehydrogenase I. Site of limited proteolytic cleavage by subtilisin.

The sequence of the first 25 residues of the homoserine dehydrogenase fragment, produced by limited proteolysis of aspartokinase I-homoserine dehydrogenase I with substilisin, has been determined. The sequence of a cyanogen bromide peptide (CB5, 59 residues), isolated from the entire protein, is also presented. Residues 1 to 18 of the subtilisin homoserine dehydrogenase fragment match the sequence 42 to 59 of peptide CB5.

Proteolytic Release of a Histidinol Dehydrogenase Fragment from the Double Enzyme Histidinol Dehydrogenase-Imidazolylacetolphosphate:l-Glutamate Aminotransferase

Abstract A double enzyme of Salmonella typhimurium, containing histidinol dehydrogenase and imidazolylacetolphosphate:l-glutamate aminotransferase, was mildly proteolyzed in an attempt to release these activities from covalent linkage. This treatment releases an active histidinol dehydrogenase fragment larger than normal histidinol dehydrogenase, while completely destroying aminotransferase activity. The normal, unfused enzymes show comparable sensitivities to proteolysis. The active fragment from the double enzyme appears to be composed of histidinol dehydrogenase subunits of roughly normal size and noncovalently bound smaller polypeptides.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K 12. 4. Isolation, molecular weight, amino acid analysis and behaviour of the sulfhydryl groups of the protein catalyzing the two activities.

The interactions between the catalytic regions of the subunit of the bifunctional enzyme aspartokinase I-homoserine dehydrogenase I, although not necessary for the catalytic functions, are shown to be of utmost importance for the regulation of the activities of the enzyme. The study of the effect of a number of –SH reagents on the protein shows that the inhibition of the homoserine dehydrogenase activity by the allosteric effector l-threonine is closely correlated with the existence of an active aspartokinase region. The kinetic study of the proteolytic reaction leading to the previously described homoserine dehydrogenase fragment indicates that a dissociation is a necessary requirement for the digestion of the aspartokinase region. Cross-linking with dimethylsuberimidate and proteolytic degradation of the fully cross-linked tetramer have been used to show that the oligomeric interactions, necessary to build the tetrameric structure, involve the kinase region only. A tentative model is proposed showing aspartokinase I-homoserine dehydrogenase I as an isologous tetramer in which the aspartokinase regions, organized as a “core”, are surrounded by the homoserine dehydrogenase regions.