Acid‐base chemical mechanism of the dihydrodipicolinate synthase from Escherichia coli

Abstract

Dihydrodipicolinate synthase (DHDPS) catalyzes the formation of dihydrodipicolinate from pyruvate and L‐aspartate‐β‐semialdehyde (ASA). The enzyme catalyzes the first committed step for the biosynthesis of L‐lysine in bacteria and plants. The enzyme from Escherichia coli is feedback inhibited by lysine, the end‐product of the pathway. Although numerous x‐ray structures have been solved for DHDPS from a number of organisms, few mechanistic and kinetic studies have been done on the enzyme. We have undertaken a study of the pH dependence of the kinetic parameters of DHDPS in order to elucidate the acid‐base chemical mechanism of the enzyme. The kinetic mechanism is ping pong with pyruvate binding to free enzyme. An active site lysine ε‐amino group attacks the carbonyl of pyruvate and forms a Schiff base intermediate. The loss of a proton from this intermediate leads to the formation of an enamine intermediate, with the loss of the proton accounting for the irreversible step and the ping pong kinetic mechanism. ASA binds to the enzyme:enamine covalent intermediate. The V/Et pH profile displays a bell shaped curve with a pKa of 7 on the acid side and a pKa of 8.6 on the basic side of the profile. The V/KASAEt pH profile is also bell shaped with pKa values of 6.5 and 8.8 on the acid and basic sides, respectively. The pKa on the acid side of the V/KASA profile likely represents the pK of the enamine intermediate and the pK on the basic side may represent the amino group of ASA. The V/KpyruvateEt pH profile displays a pKa on the basic side that likely represents the ε‐amino group of the active site lysine. Work supported by OK‐INBRE.