Monitoring enzyme catalysis in the multimeric state : Direct observation of Arthrobacter 4-hydroxybenzoyl – coenzyme A thioesterase catalytic complexes using time-resolved electrospray ionization mass spectrometry

The ability to examine real-time reaction kinetics for multimeric enzymes in their native state may offer unique insights into understanding the catalytic mechanism and its interplay with three-dimensional structure. In this study, we have used a time-resolved electrospray mass spectrometry approach to probe the kinetic mechanism of 4-hydroxybenzoyl–coenzyme A (4-HBA–CoA) thioesterase from Arthrobacter sp. strain SU in the millisecond time domain. Intact tetrameric complexes of 4-HBA–CoA thioesterase with up to four natural substrate (4-HBA–CoA) molecules bound were detected at times as early as 6 ms using an online rapid-mixing device directly coupled to an electrospray ionization time-of-flight mass spectrometer. Species corresponding to the formation of a folded tetramer of the thioesterase at charge states 16+, 17+, 18+, and 19+ around m/z 3800 were observed and assigned as individual tetramers of thioesterase and noncovalent complexes of the tetramers with up to four substrate and/or product molecules. Real-time evaluation of the reaction kinetics was accomplished by monitoring change in peak intensity corresponding to the substrate and product complexes of the tetrameric protein. The mass spectral data suggest that product 4-HBA is released from the active site of the enzyme prior to the release of product CoA following catalytic turnover. This study demonstrates the utility of this technique to provide additional molecular details for an understanding of the individual enzyme states during the thioesterase catalysis and ability to observe real-time interactions between enzyme and substrates and/ or products in the millisecond time range. 2009 Elsevier Inc. All rights reserved. Mass spectrometry has proven to be a powerful tool for the detection and study of protein–protein and protein–ligand complexes and enzyme reaction kinetics [1,2]. A common approach for characterizing the reaction kinetics for enzyme reaction pathways involves determining pre-steady-state and steady-state kinetic constants using spectrophotometric techniques (fluorescence and ultraviolet [UV]) and rapid-mixing methodologies such as stopped-flow and rapid chemical quench. However, these techniques measure the average properties of biomacromolecules or small molecules such as substrates and products. Electrospray ionization mass spectrometry (ESI–MS) is an increasingly important methodology that has grown during recent years to become a well-accepted and complementary ll rights reserved. derson). te of Basic Medical Sciences, nion Medical College, Beijing ospray ionization mass spec; ESI–TOF MS, ESI–MS with imum. technique in structural biology. Unlike spectrophotometric techniques, ESI–MS can provide detailed information about the enzymatic mechanism and reaction kinetics at the level of the quaternary protein structure [3–8]. This technique is well suited for the detection of noncovalent protein complexes [5,9–19] and their interactions with DNA [20], RNA [21], and ligands [22]. In the current investigation of the 4-hydroxybenzoyl–coenzyme A (4-HBA–CoA) thioesterase from Arthrobacter sp. strain SU, we apply quantitative time-resolved ESI–MS with time-of-flight detection (ESI–TOF MS) in the millisecond time range to monitor substrate and product occupancy of the four active sites of the native tetramer during the course of a single turnover reaction. 4HBA–CoA thioesterase catalyzes the hydrolysis of 4-HBA–CoA to 4-HBA and CoA (Scheme 1) as the final step in the bacterial 4-chlorobenzoate degradation pathway [23]. The structure of the ligated enzyme defines the location of the four substrate binding sites on the native tetramer [24]. The binding site extends from a cupshaped depression at the protein surface that binds the nucleotide moiety through a narrow tunnel that binds the panthetheine arm into an enclosed pocket that binds the hydroxybenzoyl group 210 Monitoring enzyme catalysis in multimeric state / Z. Li et al. / Anal. Biochem. 394 (2009) 209–216 (Fig. 1A). Structures of 4-HBA–CoA thioesterase homologs that hydrolyze long-chain fatty acyl–CoA thioesters differ in that the acyl binding pocket is open to solvent so as to accommodate the acyl group (Fig. 1B) [25,26]. Whereas the substrate specificity of the long-chain fatty acyl–CoA thioesterases is low [23,26], the substrate range of 4-HBA–CoA thioesterase is limited and does not include fatty acyl–CoAs [27]. The key structural unit unique to the 4HBA–CoA thioesterase is a highly extended loop-like N terminus that forms the back wall of the hydroxybenzoyl binding pocket Fig. 1. (A) Grasp-generated representation of steric and electrostatic topological features of 4-HBA–CoA binding site of Arthrobacter 4-HBA–CoA thioesterase bound with 4-hydroxyphenacyl–CoA (PDB code: 1Q4T). Ligand carbon atoms are green, oxygen atoms are red, nitrogen atoms are blue, and phosphorus atoms are orange. (B) Grasp-generated representation of steric and electrostatic topological features of long-chain fatty acyl–CoA binding site of hHTEM2 bound with undecan-2-oneCoA (PDB code: 35FO) (same coloring scheme as in panel A). (C) Stereorepresentation of backbone trace (blue) of Arthrobacter 4-HBA–CoA thioesterase dimer unit (PDB code: 1Q4T). One of the two active sites is identified by the 4-hydroxyphenacyl–CoA ligand shown in black along with the N-terminal loop shown in red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) (Fig. 1C). If the loop is fixed in position, product release must be ordered with CoA departure required to open the tunnel through which the 4-HBA can diffuse to solvent. On the other hand, if the N-terminus segment is flexible and acts simply as a flap, the 4HBA might be able to diffuse ‘‘out the back way” when the flap is open. In this case, 4-HBA release need not follow CoA release. The current work was carried out to determine whether timeresolved ESI–TOF MS could be used to determine the timing release of CoA and 4-HBA–CoA from 4-HBA–CoA thioesterase active site following catalytic turnover of substrate. In addition, it might be feasible to examine the multimeric state of the enzyme concurrent with catalysis. For this purpose, we used an online rapid-mixing device coupled directly to an ESI–TOF mass spectrometer to monitor the substrate and product ligation state of the tetramer during the single turnover reaction [4,5]. Here we present our experimental findings demonstrating that product release is indeed ordered but that 4-HBA, rather than CoA, is the first to dissociate. This result suggests that 4-HBA dissociates directly from the hydroxybenzoyl binding pocket more quickly than CoA can move out of the connecting tunnel. Moreover, we have defined the impact of the multimeric state of the enzyme in various states of ligand occupancy on catalytic turnover. Materials and methods 4-HBA–CoA thioesterase preparation Arthrobacter sp. strain SU 4-HBA–CoA thioesterase was purified as described previously [23]. The enzyme solution for analysis was prepared by overnight dialysis against 2 4 L of 10 mM ammonium acetate (pH 7.5) at 4 C. The concentration of the enzyme subunits was determined using an extinction coefficient of 23.4 mM 1 cm 1 at 280 nm. The concentrated thioesterase was diluted to 80 lM with 10 mM ammonium acetate (pH 7.5) prior to the mass spectrometric experiments. 4-HBA–CoA preparation 4-HBA–CoA was prepared according to the published procedure [23]. It was dissolved in 10 mM ammonium acetate (pH 7.5), and then metal ions such as Li, Na, and K in the solution of 4-HBA– CoA were removed using an IC-H Maxi-Clean Cartridge (Part No. 30256, Alltech, USA). The concentration of 4-HBA–CoA was determined using an extinction coefficient of 11.8 mM 1 cm 1 at 300 nm. The concentrated substrate was separately diluted to 10, 20, 30, 40, 50, and 60 lM with 10 mM ammonium acetate (pH 7.5) prior to the mass spectrometric experiments.