Prof. Julie A. Leary
|We are using ESI-FTICR MS to investigate protein-ligand noncovalent complexes. Finely controlled instrument conditions allow these ions to be transferred into the gas phase without dissociation. Proteins are mixed with their ligands in solution and the intact noncovalent complexes can be observed in the gas phase for protein-substrate, protein-inhibitor and intact multimeric protein complexes. Moreover, once these desolvated molecules are in the gas phase, important information about these complexes such as molecular weight, binding stoichiometry and relative binding strength can be determined using mass spectrometry. In particular, we are interested in the NodH sulfotransferase (NodST) from Rhizobium meliloti, which belongs to the GlcNAc6OST carbohydrate sulfotransferase family. NodST complexed with its substrate, PAPS and inhibitor, PAP were observed in the mass spectrum. Relative binding constants determined from the gas phase are close to those determined from solution. In addition, peptide mapping was used to identify the covalent sulfate enzyme intermediate formed via the random hybrid ping-pong mechanism for this enzyme.
Research is ongoing which involves using our newly developed screening assay (IEMS assay) for the analysis and identification of combinatorial libraries of inhibitors before and after incubation with immobilized enzyme. Mass spectrometric analysis of a combinatorial library of compounds which were both active and inactive towards various sulfotransferases indicates that we can identify the enzyme inhibitor compounds when eluted through a column of immobilized enzyme. Our methodology shows that inhibition is evidenced by a depletion or complete absence of ion signal in the mass spectrum obtained after reaction with the enzyme.
A variety of immobilized enzymes with combinatorial libraries exceeding 250 compounds/enzyme are currently being screened. Different immobilization techniques including reductive amination on agarose gel, metal chelation, etc, are being investigated. In general these procedures involve formation of an isourea, a diazo linkage, a peptide bond, or an alkylation reaction. Most importantly is that the immobilization method should obviously not inactivate the enzyme. Previous work in our lab has focussed on immobilizing enzymes on agarose and our results indicate that approximately 90% of the activity for each enzyme studied is retained after immobilization. The unique and advantageous facets of analyzing for inhibition using our proposed method is that very small quantities of inhibitor (low pmol) can be screened in a relatively short time frame with a large number of potential inhibitors.
A new method has been developed that allows for the determination of kinetic parameters, such as Km, Vmax, and kcat, for various enzymatic systems. The mass spectrometric method allows for study of enzymatic reactions that do not involve chromophoric changes. The technique utilizes a one point normalization factor through an internal standard for product quantification, and unlike other traditional methods, does not rely on calibration curves. Using this method, the kinetic constants have been measured for a series of sulfotransferases and phosphotransferases. This novel mass spectrometric technique has been further adapted to determine the inhibition pattern of enzyme inhibitors, measure their inhibition constants, and investigate the catalytic mechanism of bi-substrate enzymatic systems. A hybrid random Ping-Pong mechanism has been determined for NodH sulfotransferase using the ESI-MS kinetic assay, which was confirmed by the sulfated enzyme intermediate identification using ESI-FTICR-MS.
The traditional method to investigate the reaction specificity of an enzyme with different substrates is to perform individual kinetic measurements. In this case, a series of varied concentrations are required to study each substrate and a non-regression analysis program is used several times to obtain all the specificity constants for comparison. To avoid the large amount of experimental materials, long analysis time, and redundant data processing procedure involved in the traditional method, we have developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex ESI-MS assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. In our lab, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and consistency was found. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiplex substrates can be evaluated in one assay.
Using a novel ion/molecule reaction strategy, a mutase system whose substrate and product are positional isomers (as shown in the figure below) has also been successfully studied. The mass spectrometry results are identical to those obtained using traditional UV assays and are more accurate and precise. \
Our overall goals for this project are:
I. Structural Characterization of Glycosaminoglycans Using Mass Spectrometry
II. The Alpha Project
Molecular Sciences Institute link: http://www.molsci.org