Studies on the enzymes involved in the biosynthesis of cyclo-tris (N-2,3-dihydroxybenzoyl-L-seryl) in Escherichia coli: kinetic properties of the L-serine-activating enzyme

Abstract

Some kinetic properties of the formation of a thioester-bound l-seryl-enzyme intermediate are described. The rate constant of formation is 3.12 x 10(2) M(-1) s(-1) and the rate constant for spontaneous breakdown is 2.47 x 10(-3) s(-1). These constants yield a value of log K = 5.10 for the overall equilibrium constant which agrees favorably with a value of 5.20 calculated from equilibrium binding data. E(1).serine formation requires a thiol group which is extremely reactive to N-ethylmaleimide; the second-order rate constant for enzyme inactivation by this reagent is 77.1 M(-1) s(-1) at pH 6.6 and 0 C. Excess l-serine does not protect the enzyme against inactivation. In addition to an adenosine triphosphate-[(32)P]-inorganic pyrophosphate exchange, the enzyme also catalyzes an l-serine-dependent adenosine triphosphate-[(3)H]adenosine monophosphate exchange in accordance with the scheme proposed for the activation of serine.