Observation of a chemically labile, noncovalent enzyme intermediate in the reaction of metal-dependent Aquifex pyrophilus KDO8PS by time-resolved mass spectrometry.

The molecule 3-deoxy-D-manno-octulosonate (KDO) is an eight-carbon sugar that forms an integral part of lipopolysaccharide (LPS) A in the cell membranes of gram-negative bacteria.1,2 One of the enzymes involved in KDO production is 3-deoxy-D-manno-octulosonic 8-phosphate synthase (KDO8PS) which catalyzes the aldol condensation of arabinose-5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form inorganic phosphate (Pi) and a precursor to KDO, 3-deoxy-D-manno-octulosonic 8-phosphate (KDO8P). A number of KDO8P synthases from different organisms have been identified and characterized.2–6 These studies have revealed two distinct classes of KDO8PS: those that do not require a divalent metal ion for activity (metal-independent) and those that do (metal-dependent). The former includes KDO8PS from various E. coli strains, the latter includes KDO8PS from Helicobacter pylori (a human gastric pathogen) and the thermophilic organisms Aquifex aeolicus and Aquifex pyrophilus. Although the overall amino acid sequence conservation is low (30%) between different KDO8PS members, the crystal structures reveal that the folds and active site residues of the two classes are highly conserved. The exception is a cysteine in the metal- dependent enzymes that replaces an asparagine in the metal-independent enzymes.1,7–9 The cysteine coordinates the metal, and it has been shown that the reciprocal mutations alter the metal-dependence of both classes of KDO8PS.10 Structural and mechanistic studies have proposed that the metal facilitates proper orientation of substrates in the active site and/or facilitates communication between the four subunits that make up the homotetrameric KDO8PS.1,2,8,11,12 Detailed mechanistic information for the KDO8PS reaction has been obtained from the analysis of the pre-steady state reaction kinetics of metal-dependent and -independent KDO8PS using rapid chemical quenching and radiolabeled substrates. For all KDO8PS, product release is the rate-limiting step in the reaction, with the rate of chemistry ranging from 110 s–1 (E. coli at 25°C), to ~200 s–1 (A. pyrophilus at 60°C).6,10,11 Early mechanistic work on E. coli KDO8PS proposed that the reaction proceeded through the formation of a noncovalently bound, acyclic hemiketal phosphate intermediate ([I], Fig. 1).11 However, with no distinct spectroscopic signal, a short lifetime, and chemical lability, direct observation of this intermediate was expected to be difficult. Figure 1 Reaction of A5P with PEP proceeds through the formation of an acyclic hemiketal intermediate [I].11 Since the development of electrospray ionization (ESI), mass spectral analysis of biological molecules has become routine, with increasing evidence suggesting that physiologically relevant structures are maintained in the gas phase. This is supported by numerous studies of noncovalent protein–protein and protein–small molecule complexes.13,14 Coupled with its high sensitivity and accuracy, several groups recognized the potential for kinetic and mechanistic analysis of enzymatic reactions by mass spectrometry. This has included both direct and offline monitoring of reactions.15–22 In particular, following reactions in the pre-steady state regime holds the greatest potential for identifying enzyme intermediates which are formed transiently in the active site.23 To examine pre-steady-state kinetics directly, millisecond time resolution is often required, and a few different experimental setups for this type of analysis have been described.24–27 Our laboratory developed a rapid-mixing attachment for online analysis of enzymatic reactions with a theoretical time resolution of 7 ms. The principles of the design are simple: syringes containing protein and substrate solutions are driven into a mixing tee by a syringe pump. The mixing tee is connected to a capillary (the reaction vessel) which terminates at the ESI source. A number of control experiments have shown that the kinetic constants derived from online rapid-mixing mass spectrometry vs. solution techniques are the same, indicating that quantitative rates may be obtained from online rapid-mixing mass spectrometry.28,29 After careful optimization of gas pressures, temperatures, and capillary exit voltages, this mixing apparatus was used to detect the formation of a transient intermediate in the E. coli KDO8PS reaction under single turnover conditions, confirming the presence of a noncova-lent enzyme:intermediate complex with a mass consistent with the acyclic hemiketal phosphate. This provided the first direct evidence for this transient intermediate in the metal-independent class of KDO8PS.29 Although a recent crystal structure of a triple mutant of metal-dependent A. aeolicus KDO8PS revealed electron density in the active site consistent with the proposed reaction intermediate, direct detection of the intermediate under native conditions has not been achieved.1,7–9,12 In this study we observed the proposed hemiketal phosphate intermediate in the metal-dependent KDO8PS reaction from Aquifex pyrophilus under single turnover conditions with time-resolved mass spectrometry. Distinct PEP, KDO8P, intermediate, and Pi species in noncovalent complexes with Cd2+-bound KDO8PS are observed, supporting the broad applicability of time-resolved mass spectrometry as a tool for mechanistic studies of metalloenzymes.