[Purification, molecular multiplicity and kinetic properties of "biosynthetic" L-threonine dehydratase from E. coli K-12].
"Biosynthetic" L-threonine dehydratase was purified to homogeneous state with yield 29% of total activity from E. coli K-12. The cells were disrupted by means of ultra sound. Nucleic acids and nucleoproteins were precipitated with protamine sulphate, the proteins were fractioned with (NH4)2SO4, by gel filtration through Sephadex G-25 followed by chromatography on DEAE-cellulose using stepways elution by changing the pH-values. The homogenity of the enzyme was shown by polyacrylamide gel disc electrophoresis in the presence of dodecylsulphate. The enzyme consists of equal subunits having a molecular weight about 57000. The polyacrylamide gel disc electrophoresis had shown that the native enzyme consists of a set of oligomeric forms. The multiplisity of molecular organization of the enzyme was relfected in complicated kinetic behavior: at pH greater than 9 on the plots of initial reaction rate (upsilon) versus initial substrate concentration ([S]0) there were four inflexion points (two intermediate plateaux) the position and deepness of which depended on enzyme concentration. Kinetic properties of the highly purified enzyme and the enzyme in crude cell extracts at pH 9.3 and 7.4 were identical. At pH 8,3 on the upsilon versus [S]0 plots appeared two inflexion points (one intermediate plateau), 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 had shown that oligomeric forms underwent a slow polymerization. It is 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 upsilon versus [S]0 plots. The rate of equilibrium between the oligomeric forms is small in comparison with the enzyme reaction velosity, that lead to the complex kinetic curves appearing as a result of summing up the kinetics inherent to the individual forms.
D004926:Escherichia coli / Q000201:enzymology*; D006836:Hydro-Lyases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D013913:Threonine Dehydratase / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D006836:Hydro-Lyases; D013913:Threonine Dehydratase
Ochistka, mnozhestvennost' molekuliarnykh form i kineticheskie svoĭstva "biosinteticheskoĭ" L-treonindegiratazy iz bakteriĭ E. coli K-12