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Threonine Deaminase from Escherichia coli

1973; Elsevier BV; Volume: 248; Issue: 10 Linguagem: Inglês

10.1016/s0021-9258(19)43959-8

1083-351X

David H. Calhoun, Ronald A. Rimerman, G. Wesley Hatfield,

Hemoglobin structure and function

Abstract A procedure has been devised for the purification of the biosynthetic l-threonine deaminase (l-threonine hydrolase, deaminating: EC 4.2.1.16) from a genetically derepressed mutant, strain TIR8, of Escherichia coli K-12. The procedure produces a purification of 400-fold relative to derepressed enzyme levels and 4000-fold relative to repressed levels. The purity of the final enzyme preparation has been determined by sodium dodecyl sulfate gel electrophoresis and by sedimentation equilibrium experiments. The native enzyme, molecular weight 204,000, is a tetramer composed of four apparently identical subunits, molecular weight 51,000. When the native enzyme is resolved of its cofactor, pyridoxal 5'-monophosphate, catalytically inactive apodimers, molecular weight 102,000, are formed. Addition of pyridoxal 5'-monophosphate to the apodimers promotes reassociation to catalytically active tetramers. The kinetic data of the l-threonine deaminase reaction are similar with the pure enzyme and with partially purified preparations. l-Isoleucine is a competitive allosteric inhibitor. The enzyme exhibits cooperative homotropic interactions with respect to the substrate only in the presence of l-isoleucine. These interactions are reversed by l-valine. The enzyme exists in a pH-dependent equilibrium between two distinct and catalytically active species; a tetramer sensitive to l-isoleucine inhibition and a dimer insensitive to l-isoleucine inhibition. The tetramer is favored at pH values from 6.0 to 7.5 while at pH values from 8.5 to 10.0 the enzyme is present almost exclusively as the dimer. At pH 8.0 a mixture of the two is present.