Evidence for Enzyme-bound β-Aspartyl Phosphate in the Aspartokinase Reaction

Abstract

The lysine-sensitive aspartokinase from Escherichia coli B has been purified to a nearly homogenous state.When incubated with substrates (ATP, aspartate, and Mg++) and then subjected to gel atration on Sephadex G-50, a complex containing approximately 0.8 mole of aspartate and 0.8 mole of phosphate (from the y-phosphate of ATP) per 100,000 g of protein can be isolated.This complex is unstable, having a half-life of about 26 min at 25' and pH 7.0.When treated with hydroxylamine, the complex yields products electrophoretically identical with B-aspartylhydroxamate and aspartic acid.When the complex is incubated with ADP and Mg+f, ATP is formed.Lysine, ADP, and excess Mg+f, all of which inhibit aspartokinase activity, also decrease the measurable amount of complex formed.Aspartokinase (ATP-n-aspartate4-phospbotransferase, EC 2.7.2.4) participates in a common biochemical reaction sequence, the activation of a carboxyl group prior to its participation in either a reductive or a group transfer reaction.Several enzymes catalyzing group transfer reactions coupled to ATP cleavage may involve bound acyl phosphates as intermediates (2).If bound acyl phosphate is favorable for such reactions, it may also be favorable for other reaction sequences in which the activation and reduction or transfer are catalyzed by separate enzymes.This seems especially intriguing if one thinks of the multitudinous enzyme systems in the so-called "soluble" portion of the cell as having some degree of macromolecular organization.The various aspartokinases of Escherichia coli have been thor-