Kinetic Analysis of NodST Sulfotransferase Using an Electrospray Ionization Mass Spectrometry Assay

Pi, Na; Armstrong, Joshua I.; Bertozzi, Carolyn R.; Leary, Julie A.

doi:10.1021/bi020457g

Cited by 57 publications

(64 citation statements)

References 28 publications

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“…R can be obtained by analyzing a mixture of the internal standard and the product of known concentration [27][28][29].…”

Section: Individual Kinetic Measurementsmentioning

confidence: 99%

“…The development of soft ionization techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption (MALDI), has made mass spectrometry an excellent complementary technique to conventional spectrophotometric methods for studying enzyme kinetics [23][24][25][26][27][28][29]. With the improvements of resolving power, sensitivity, and versatility, mass spectrometry has become a highly competitive analytical method for detection and characterization of biochemical reaction intermediates that can be used to elucidate enzymatic reaction pathways and catalytic mechanisms [16 -22].…”

mentioning

confidence: 99%

“…Likewise, Leary and coworkers reported the identification of a sulfated NodH sulfotransferase (NodST) intermediate formed in a hybrid Ping-Pong mechanism using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry [17]. A facile and broadly applicable ESI-MS assay developed by Leary and coworkers has been used for kinetic analyses on several enzyme systems such as glutathione S-transferase (GST) [27], hexokinase [28], and NodH sulfotransferase (NodST) [29]. The mass spectrometry assay is especially useful because traditional spectrophotometric assays are not applicable to NodST kinetics since no shift in absorption accompanies sulfuryl group transfer.…”

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confidence: 99%

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Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Leary

2004

J. Am. Soc. Mass Spectrom.

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The traditional method used 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 procedures 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 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. The system investigated was the bacterial sulfotransferase NodH (NodST), which is a host specific nod gene product that catalyzes the sulfate group transfer from 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS) to natural Nod factors or synthetic chitooligosaccharides. Herein, 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 found to be consistent. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiple substrates can be evaluated in one assay. (J Am Soc Mass Spectrom 2004, 15, 233-243)

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“…R can be obtained by analyzing a mixture of the internal standard and the product of known concentration [27][28][29].…”

Section: Individual Kinetic Measurementsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Leary

2004

J. Am. Soc. Mass Spectrom.

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“…In the past decade, considerable effort was invested to develop MS-based detection schemes capable of assaying inhibition of enzyme-mediated reactions [15,16]. The use of electrospray ionization (ESI)-MS is rapidly expanding into the study of enzyme kinetics [17][18][19][20][21][22][23]. Also, combinations of chromatographic and electrophoretic separation methods with ESI-MS, such as liquid chromatography (LC)/MS [24], LC/tandem mass spectroscopy (MS/MS) [25], gas chromatography (GC)/MS [26], frontal affinity chromatography (FAC)/MS [27], or capillary electrophoresis (CE)/MS [28] have been reported for the determination of acetylcholine or the screening of AChE inhibitors.…”

mentioning

confidence: 99%

Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

Yao

Wei

et al. 2008

J. Am. Soc. Mass Spectrom.

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A matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS)-based assay was developed for kinetic measurements and inhibitor screening of acetylcholinesterase. Here, FTMS coupled to MALDI was applied to quantitative analysis of choline using the ratio of choline/acetylcholine without the use of additional internal standard, which simplified the experiment. The Michaelis constant (K m ) of acetylcholinesterase (AChE) was determined to be 73.9 mol L Ϫ1 by this approach. For Huperzine A, the linear mixed inhibition of AChE reflected the presence of competitive and noncompetitive components. The half maximal inhibitory concentration (IC 50 ) value of galantamine obtained for AChE was 2.39 mol L Ϫ1 . Inhibitory potentials of Rhizoma Coptidis extracts were identified with the present method. In light of the results the referred extracts as a whole showed inhibitory action against AChE. The use of high-resolution FTMS largely eliminated the interference with the determination of ACh and Ch, produced by the low-mass compounds of chemical libraries for inhibitor screening. The excellent correlation with the reported kinetic parameters confirms that the MS-based assay is both accurate and precise for determining kinetic constants and for identifying enzyme inhibitors. The obvious advantages were demonstrated for quantitative analysis and also high-throughput characterization. This study offers a perspective into the utility of MALDI-FTMS as an alternate quantitative tool for inhibitor screening of

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“…Recently, the development of soft ionization techniques, such as electrospray ionization (ESI), has made MS an excellent complementary technique to conventional methods for studying enzyme kinetics (24)(25)(26)(27)(28)(29)(30). A facile and broadly applicable ESI͞MS assay developed by Leary and coworkers has been implemented for kinetic analyses and substrate specificity evaluation of several enzyme systems such as glutathione S-transferase (26), hexokinase (27), phosphoglucomutase (28), and NodH sulfotransferase (29,30). Herein, a somewhat different ESI͞MS technique was used to develop assays for NovL and NovM individually and in tandem.…”

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confidence: 99%

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton

Meyers

Pacholec

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The tripartite scaffold of the natural product antibiotic novobiocin is assembled by the tandem action of novobiocin ligase (NovL) and novobiocic acid noviosyl transferase (NovM). The noviosyl ring of the tripartite scaffold is further decorated by a methyltransferase (NovP) and a carbamoyltransferase (NovN), resulting in the formation of novobiocin. To facilitate kinetic evaluation of alternate substrate usage by NovL and NovM toward the creation of variant antibiotic scaffolds, an electrospray ionization͞MS assay for obtaining kinetic measurements is presented for NovL and NovM separately, in each case with natural substrate and the 3-methyl-4-hydroxybenzoic acid analog. Additionally, assays of tandem two-enzyme (NovL͞NovM) and three-enzyme (NovL͞NovM͞NovP) incubations were developed. The development of these assays allows for the direct detection of each intermediate followed by its utilization as substrate for the next enzyme, as well as the subsequent formation of final product as a function of time. This MS tandem assay is useful for optimization of conditions for chemoenzymatic generation of novobiocin and is also suitable for evaluation of competitive usage of variant substrate analogs by multiple enzymes. The studies presented here serve as a platform for the subsequent expansion of the repertoire of coumarin-based antibiotics.

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Kinetic Analysis of NodST Sulfotransferase Using an Electrospray Ionization Mass Spectrometry Assay

Cited by 57 publications

References 28 publications

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Determination of enzyme/substrate specificity constants using a multiple substrate ESI-MS assay

Monitoring enzyme reaction and screening of inhibitors of acetylcholinesterase by quantitative matrix-assisted laser desorption/ionization fourier transform mass spectrometry

Mass spectrometric characterization of a three-enzyme tandem reaction for assembly and modification of the novobiocin skeleton

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