Purification of biosynthetic threonine deaminase from Escherichia coli

Abstract

Biosynthetic threonine deaminase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) was purified to apparent homogeneity from cell extracts of Escherichia coli by chromatographic procedures using valine-Sepharose, isoleucine- N-hexamethyleneamine-Sepharose and hydroxyapatite with an overall yield of 40%. Analytical ultracentrifugation shows a molecular weight of 214 000. In sodium dodecyl sulfate gel electrophoresis, the enzyme migrates as a single band corresponding to a molecular weight of about 50 000. These data confirm that the enzyme is a tetramer. The sedimentation coefficient, s 0 2 0, w , determined by differential sedimentation experiments is 9.2 S. The enzyme shows absorption maxima at 415 and 280 nm. Determination of pyridoxal phosphate by three independent methods shows the presence of two molecules of pyridoxal phosphate per enzyme molecule, the different methods being in excellent agreement. Equilibrium dialysis experiments establish the presence of two isoleucine binding sites. The Scatchard plot suggests non-cooperativity of these sites. The association constant for isoleucine is 1.2 · 10 5 M −1.