Probing the role of tightly bound phosphoenolpyruvate in Escherichia coli 3-deoxy- d- manno-octulosonate 8-phosphate synthase catalysis using quantitative time-resolved electrospray ionization mass spectrometry in the millisecond time range

Escherichia coli 3-deoxy- d- manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) and d-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. The enzyme is often isolated with varying amounts of tightly bound PEP substrate. To better understand the role of tightly bound PEP in E. coli KDO8P synthase catalysis, a combination of transient kinetic methodologies including rapid chemical quench and mass spectrometry techniques such as time-resolved electrospray ionization mass spectrometry (ESI-TOF MS) were used to study the enzyme purified both in the PEP-bound state and in the unbound state. Pre-steady state burst and single-turnover experiments using radiolabeled [1- 14C] and [ 32P]A5P revealed significant kinetic differences between these enzyme preparations. The active sites concentrations for the bound and unbound states of the enzyme were almost the same (∼100%) and the product release for both states of the enzyme was rate limiting. However, the rate constant of product formation for the PEP-bound enzyme (125 s −1) was higher than that of the unbound enzyme (46 s −1). This was further confirmed by single-turnover experiments using radiolabeled [ 32P]A5P. Interestingly, when PEP was removed from the PEP-bound enzyme and external PEP was added before the kinetic experiments, both the pre-steady state burst and the single-turnover kinetic parameters were similar to those of the enzyme purified in the unbound state. The rate constants of product formation were determined as 44 s −1 (burst experiment) and 48 s −1 (single-turnover experiment). The reaction kinetics of the E. coli KDO8P synthase was also followed by time-resolved ESI mass spectrometry. To validate the suitability of this technique for conducting enzyme kinetics, the standard reaction of p-nitrophenyl acetate hydrolysis by chymotrypsin was analyzed by stopped-flow and time-resolved ESI-TOF MS. The rate constant of p-nitrophenol formation followed by stopped-flow spectrophotometry matched perfectly the rate constant of acetyl–chymotrypsin intermediate formation followed by time-resolved ESI-TOF MS (0.1 s −1). The catalytic properties of the PEP-bound and unbound states of the E. coli KDO8P synthase were then studied on a millisecond time scale. The changes in the intensity of E•PEP, E•KDO8P, and E•intermediate complexes as a function of time were quantified and the reaction kinetics were modeled using KinTekSim simulation softwar