Studies of homogeneous "biosynthetic" L-threonine dehydratase from Escherichia coli K-12. Some kinetic properties and molecular multiplicity.
"Biosynthetic" L-threonine dehydratase (EC 4.2.1.16) was purified to a homogeneous state with 29% yield of total activity from Escherichia coli K-12. The homogeneity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecyl sulphate. The enzyme consisted of equal subunits having a molecular weight of about 57 000. The polyacrylamide gel disc electrophoresis has shown that the native enzyme consisted of a set of oligomeric forms. The multiplicity of molecular organization of the enzyme was reflected in complicated kinetic behaviour: at pH greater than 9 on the plots of initial reaction rate (v) versus initial substrate concentration ([S]o) there were four inflexion points (two intermediate plateaux), the position and deepness of which depended on enzyme concentration. At pH 8.3 on the v versus [S]o plots appeared two inflexion points (one intermediate plateu), the position of which practically did not depend on enzyme concentration in the reaction mixture, but strongly depended on the enzyme concentration in the stock solution. Repeated polyacrylamide gel disc electrophoresis of several oligomeric forms, isolated by the first electrophoresis, has shown that the oligomeric forms underwent a slow polymerization. It was suggested that "biosynthetic" L-threonine dehydratase from E. coli K-12 is a set of multiple oligomeric forms, having different kinetic parameters. Probably, each form of the enzyme has a "simple" kinetics characterized by hyperbolic or sigmoidal shape of v versus [S]o plots. The rate of equilibrium installation between the oligomeric forms was small in comparison with the enzyme reaction velocity, that lead to the complex kinetic curves, appearing as a result of summing up of the kinetics inherent to theindividual forms.
D004926:Escherichia coli / Q000201:enzymology*; D006836:Hydro-Lyases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013912:Threonine / Q000494:pharmacology; D013913:Threonine Dehydratase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D013912:Threonine; D006836:Hydro-Lyases; D013913:Threonine Dehydratase