Kinetic Characterization of Enzyme Inhibitors Using Electrospray

Nov 14, 2001 - Crump Institute for Molecular Imaging, Department of Molecular and Medical ... of Psychiatry & Biobehavioral Sciences, The Neuropsychia...
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Anal. Chem. 2001, 73, 6024-6029

Kinetic Characterization of Enzyme Inhibitors Using Electrospray-Ionization Mass Spectrometry Coupled with Multiple Reaction Monitoring Andrew J. Norris,†,‡ Julian P. Whitelegge,§ Kym F. Faull,*,§ and Tatsushi Toyokuni*,†

Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095-1770, Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569, and Department of Psychiatry & Biobehavioral Sciences, The Neuropsychiatric Institute, and The Pasarow Mass Spectrometry Laboratory, University of California, Los Angeles, California 90095-1569

Electrospray ionization mass spectrometry coupled to multiple reaction monitoring (ESI-MS/MRM) has been applied for the first time to analyze enzyme inhibitor kinetics. Specifically, a known competitive inhibitor, guanosine 5′-monophosphate (GMP), and a synthetic, transition-state analogue inhibitor, guanosine 5′-[1D-(1,3,4/2)5-methyl-5-cyclohexene-1,2,3,4-tetrol 1-diphosphate] (1) have been characterized against recombinant fucosyltransferase (Fuc-T) V using ESI-MS/MRM. Dixon analysis with GMP yielded a signature plot for competitive inhibition. Nonlinear regression analysis gave a Ki of 211.8 ( 24.7 µM. The conventional analysis using GDP-[U-14C]Fuc yielded a similar Ki value of 235.6 ( 59.4 µM, confirming the validity of the MS-based method. The synthetic inhibitor 1 showed potent competitive inhibition with a Ki of 25.6 ( 2.8 µM. Although 1 possesses a chemically reactive allyl phosphate group, ESI-MS/MRM showed that there was no reduction in the concentration of 1 and no production of a predicted metabolite GDP during the assay. MS/MS also confirmed the absence of a possible pseudo-trisaccharide product. The results clearly show that 1 is neither a slow-reacting donor nor does it act as a suicide-type inhibitor toward Fuc-T V. ESIMS/MRM is therefore a powerful tool for the kinetic characterization of enzyme inhibitors, providing complete disclosure of the mechanism of action of 1 as an inhibitor. The characterization of enzyme inhibitors is a crucial part of biochemical and biomedical research allowing a fundamental understanding of how an inhibitor (or drug) interacts with an enzyme. The role of mass spectrometry (MS) in the characterization of enzyme inhibitors has been expanding rapidly. Some of the current contributions include the characterization of the interface structure of enzyme-inhibitor complexes in conjunction with the hydrogen-deuterium exchange method,1 the detection * To whom correspondence should be addressed. T.T.: (e-mail) ttoyokuni@ mednet.ucla.edu; (fax) (310) 206-8975. K.F.F.: (e-mail) [email protected]; (fax) (310) 206-2161. † Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, UCLA School of Medicine. ‡ Department of Chemistry and Biochemistry. § Department of Psychiatry & Biobehavioral Sciences, The Neuropsychiatric Institute, and The Pasarow Mass Spectrometry Laboratory.

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of noncovalent complexes between an enzyme and its inhibitor,2,3 investigation of covalent enzyme intermediates,4-6 and the rapid screening of libraries of potential enzyme inhibitors.7-9 These mass spectrometric techniques are important for the characterization of the physical interaction in solution between the enzyme and its inhibitor. Since the first reported application of MS for the analysis of enzyme kinetics by Henion’s group in 1995,10 there have been considerable advances of MS into the areas of enzymology.11-15 Thus, it is of particular interest to extend this technology into the kinetic characterization of inhibitors. This report will demonstrate the application of electrospray ionization mass spectrometry coupled to multiple reaction monitoring (ESI-MS/MRM) to characterize inhibitors of the enzyme fucosyltransferase (Fuc-T) V. The investigation of Fuc-T V inhibitors has led to important findings regarding the proposed mechanism of this enzyme.16-18 Previously, guanosine 5′-[1D(1,3,4/2)-5-methyl-5-cyclohexene-1,2,3,4-tetrol 1-diphosphate] (1) was synthesized as an unsaturated carbocyclic analogue of the (1) Akashi, S.; Takio, K. Protein Sci. 2000, 9, 2497-2505. (2) Douglas, D. J.; Collings, B. A.; Numao, S.; Nesatyy, V. J. Rapid Commun. Mass Spectrom. 2001, 15, 89-96. (3) Wang, Y.; Schubert, M.; Ingendoh, A.; Franzen, J. Rapid Commun. Mass Spectrom. 2000, 14, 12-17. (4) Przybylski, M.; Glocker, M. O.; Nestel, U.; Schnaible, V.; Blu ¨ ggel, M.; Diederichs, K.; Weckesser, J.; Schad, M.; Schmid, A.; Welte, W.; Benz, R. Protein Sci. 1996, 5, 1477-1489. (5) Bordini, E.; Hamdan, M. Rapid Commun. Mass Spectrom. 1999, 13, 11431151. (6) Bakhtiar, R.; Leung, K. H. Rapid Commun. Mass Spectrom. 1997, 11, 19351937. (7) Wu, J.; Takayama, S.; Wong, C. H.; Siuzdak, G. Chem. Biol. 1997, 4, 653657. (8) Takayama, S.; Martin, R.; Wu, J. Y.; Laslo, K.; Siuzdak, G.; Wong, C. H. J. Am. Chem. Soc. 1997, 119, 8146-8151. (9) Cancilla, M. T.; Leavell, M. D.; Chow, J.; Leary, J. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12008-12013. (10) Hsieh, F. Y.; Tong, X.; Wachs, T.; Ganem, B.; Henion, J. Anal. Biochem. 1995, 229, 20-25. (11) Bothner, B.; Chavez, R.; Wei, J.; Strupp, C.; Phung, Q.; Schneemann, A.; Siuzdak, G. J. Biol. Chem. 2000, 275, 13455-13459. (12) Zechel, D. L.; Konermann, L.; Withers, S. G.; Douglas, D. J. Biochemistry 1998, 37, 7664-7669. (13) Gross, J. W.; Hegeman, A. D.; Vestling, M. M.; Frey, P. A. Biochemistry 2000, 39, 13633-13640. (14) Houston, C. T.; Taylor, W. P.; Widlanski, T. S.; Reilly, J. P. Anal. Chem. 2000, 72, 3311-3319. (15) Norris, A. J.; Whitelegge, J. P.; Faull, K. F.; Toyokuni, T. Biochemistry 2001, 40, 3774-3779. 10.1021/ac015574g CCC: $20.00

© 2001 American Chemical Society Published on Web 11/14/2001

normal substrate GDP-L-fucopyranose (GDP-Fuc).19 The structure of 1 was designed to mimic the proposed flattened oxocarbenium ion-like conformation of the fucosyl residue in the transition state. This oxocarbenium ion-like transition state is important for many glycosidase,20,21 glycosyltransferase,22,23 and other enzymatic reactions.22,24 Indeed, preliminary experiments focusing on the effects of 1 on enzyme activity indicated that the inhibitory potential against a crude preparation of R(1f3/4)Fuc-T was significant.19 However, detailed kinetic characterization of 1 has yet to be reported. We previously reported the usefulness of ESI-MS/MRM in the analysis of enzyme kinetics.15 Here we have extended the ESI-MS/MRM methodology to the kinetic characterization of the Fuc-T inhibitor 1 and include a demonstration of the flexibility of the technique by monitoring the chemical state of 1 during the Fuc-T-catalyzed reaction. EXPERIMENTAL SECTION Chemical and Reagents. Soluble recombinant human Fuc-T V, GDP-Fuc (sodium salt), Lewis-X trisaccharide (Galβ1-4(FucR13)GlcNAcβ1-R: Lex trisaccharide), and N-acetyllactosamine (Galβ14GlcNAc: LacNAc) were purchased from Calbiochem (San Diego, CA.). Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (BisTris), 2′-deoxyguanosine 5′-diphosphate (dGDP) (sodium salt), guanosine 5′-diphosphate (GDP) (sodium salt), and guanosine 5′monophosphate (GMP) (sodium salt) were purchased from Sigma Aldrich (St Louis, MO). GDP-[U-14C]Fuc (287 mCi/mmol) was purchased from Amersham Pharmacia Biotech. Quartz distilled water (>16 mΩ cm-1) was produced in-house, and all other reagents and solvents were of analytical grade. The inhibitor 1 was synthesized as previously described.19 The compound was lyophilized as a disodium salt and was >99% pure by 1H NMR. Enzyme Reactions. The reactions were carried out as previously described.15 Briefly, each reaction tube contained 20 mM Bis-Tris (pH 6.8), 10 mM MnCl2, 2 mM dithiothreitol (DTT), and 20 mM LacNAc. For the inhibition analysis with GMP, GDP-Fuc was held constant at 0.05 or 0.15 mM and GMP was varied at 0.00, 0.25, 0.50, 1.00, 2.00, and 3.00 mM. For the inhibition analysis with 1, GDP-Fuc was held constant at 0.05, 0.15, and 0.20 mM and the concentration of 1 was varied at 0.00, 0.02, 0.05, 0.10, 0.20, and 0.30 mM. All reactions had a total volume of 49.15 µL. Before use, the enzyme was diluted 5-fold with a solution containing 10 mM DTT, 10 mM Bis-Tris, and 10 mM MnCl2. Reactions were started by the addition of 0.085 munit of Fuc-T V (0.85 µL of the diluted enzyme preparation) and incubated at 37 °C for 0, 6, 12, and 18 min. Control reactions were carried out in the absence of both enzyme and GDP-Fuc. Reactions were (16) Murray, B. W.; Takayama, S.; Schultz, J.; Wong, C. H. Biochemistry 1996, 35, 11183-11195. (17) Murray, B. W.; Wittmann, V.; Burkart, M. D.; Hung, S. C.; Wong, C. H. Biochemistry 1997, 36, 823-831. (18) Qiao, L.; Murray, B. W.; Shimazaki, M.; Schultz, J.; Wong, C. H. J. Am. Chem. Soc. 1996, 118, 7653-7662. (19) Cai, S.; Stroud, M. R.; Hakomori, S.; Toyokuni, T. J. Org. Chem. 1992, 57, 6693-6696. (20) Werner, R. M.; Stivers, J. T. Biochemistry 2000, 39, 14054-14064. (21) Davies, G. J.; Mackenzie, L.; Varrot, A.; Dauter, M.; Brzozowski, A. M.; Schu ¨ lein, M.; Withers, S. G. Biochemistry 1998, 37, 11707-11713. (22) Campbell, R. E.; Mosimann, S. C.; Tanner, M. E.; Strynadka, N. C. Biochemistry 2000, 39, 14993-15001. (23) Horenstein, B. A.; Bruner, M. J. Am. Chem. Soc. 1998, 120, 1357-1362. (24) He´roux, A.; White, E. L.; Ross, L. J.; Kuzin, A. P.; Borhani, D. W. Structure 2000, 8, 1309-1318.

terminated by addition of 10 µL of the reaction mixture into a tube containing 30 µL of 70% aqueous MeOH and 0.5 nmol of dGDP as an internal standard. Each sample was centrifuged (20000g, 10 min), 15 µL was removed and diluted with 135 µL of MeCN/H2O/Et3N (35/65/0.2, v/v/v) (AWT), and aliquots (20 µL) were analyzed by ESI-MS. Radioactive Inhibition Assay. The conditions and procedures were the same as described above for GMP with the following exceptions. The concentration of substrate, GDP-[U-14C]Fuc (3.6 mCi/mmol), was 0.15 mM. For each time point, 10 µL was removed to be terminated in a tube containing 20 µL of MeOH, diluted with 500 µL of H2O, applied to a 1-mL Dowex 1-X8 column (Cl-), and washed with H2O (3 × 0.4 mL) as described.18 The flow-through and washings were collected in 10 mL of ScintiVerse I scintillation cocktail (Fisher, Los Angeles, CA), and the radioactivity content was measured by an ISOCAP/300 liquid scintillation spectrometer (Searle Analytic Inc. Des Plaines, IL). ESI-MS/MRM. A Perkin-Elmer Sciex (Thornhill, Canada) API III triple quadrupole mass spectrometer was tuned and calibrated in the positive ion mode as previously described.15 Under standard resolution conditions, the isotopes of the poly(propylene glycol)/ NH4+ singly charged ion at m/z 906 were resolved with a 40% valley. Under degraded resolution conditions (to enhance sensitivity), the isotopes at m/z 906 were not resolved from one another. For the analysis of the enzyme reaction mixtures under the degraded resolution conditions in the negative ion mode, the polarity of the instrument was reversed, the orifice was set to -85 V, and a stream of AWT was constantly infused into the ion source at 22 µL/min. Reaction mixture samples (dissolved in AWT at 20 pmol/µL) were injected into this stream via a 20-µL injection loop. Normal spectra were recorded by scanning from m/z 300 to 2200 (0.3-Da step size, 6.66 s/scan). Fragment ion spectra of Q1 preselected parent ions were recorded by scanning Q3 from m/z 50 to 600 (step size 0.3 Da, 7.43 s/scan). Fragment ion spectra and MRM recordings were made with 10% nitrogen in argon collision gas with a collision gas thickness (CGT) instrument setting of 120 and a rod offset (R0-R2) of 30 V as previously described.15 For recording the reaction kinetics, the relative intensity of the transition of the corresponding (M - H)- ion to the most abundant MS/MS fragment ion was monitored during the course of the reaction. Up to eight transitions were typically monitored for each analysis (GDP, m/z 442 f 159; GDP, m/z 442 f 150; dGDP, m/z 426 f 159; dGDP, m/z 426 f 150; GDPFuc, m/z 588 f 424; 1, m/z 584 f 301; 1, m/z 584 f 362; 1, m/z 584 f 424) with a total scan time of 2.3 s which yielded 40-50 data points for each transition from each sample injected. From the three transitions arising from m/z 584, the most abundant signal originated from the transition 1 m/z 584 [(1 H)-] f 301 [(C7H11O9P2)-] and this was used to monitor the inhibitor during the reaction progress. The enzyme activity and data analysis was calculated as previously described.15 ESI-MS and MS/MS Spectra of 1. Using AWT as solvent, the negative ion ESI spectra and MS/MS spectra of 1 were recorded with the identical collisionally activated dissociation condition as previously reported for GDP, GDP-Fuc, and dGDP.15 Standard Curve. A standard curve was constructed from the data obtained from a series of samples consisting of 2.00, 1.00, 0.67, and 0.05 nmol of GDP placed into tubes containing 30 µL of Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

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Figure 1. MS and MS/MS data for 1. (A) Negative ion mode ESI-MS spectra of the disodium salt of 1 using ATW as a solvent. Under these conditions, the predominant signal was the singly charged alkali metal-free ion (M - H)- at m/z 583.9 (calcd 584.1 Da) with ∼10% as the sodium adduct (m/z 605.8). The lower m/z are unknown. (B) Negative ion mode MS/MS spectra of 1 showing the structurally significant fragment ions: (PO3)- at m/z 79.4 (calcd 79.0 Da), (HO6P2)- at m/z 158.9 (calcd 158.9 Da), (C7H11O9P2)- at m/z 300.8 (calcd 300.9 Da), (C10H11N5O7P)at m/z 344.9 (calcd 344.0 Da), (C10H13N5O8P)- at m/z 361.9 (calcd 362.1 Da), (C10H12N5O10P2)- at m/z 423.8 (calcd 424.0 Da), and (C10H14N5O11P2)- at m/z 442.0 (calcd 442.0 Da). The most abundant fragment ion (C7H11O9P2)- at m/z 300.8 was chosen to monitor the chemical stability of 1 in the presence of Fuc-T V. The parent ion is indicated by (P).

70% MeOH and 0.25 nmol of dGDP. The reaction mixture (10 µL) devoid of LacNAc was added to each tube, and each sample was centrifuged for ESI-MS analysis as described above. Data Processing. Representative spectra were computed as the average of all the spectra accrued from each injection of a standard using instrument-supplied software (MacSpec, version 3.3, PE Sciex, Ontario, Canada). For the measurement of the MRM responses, the profiles were smoothed 10 times and the relative peak areas were measured after exporting the data to the IGOR Pro computer program (version 3, WaveMetrics, Onc., Lake Oswego, OR). To obtain units of velocity, the peak areas arising from the reaction product transition m/z 442 [(GDP - H)-] f 159 [(P2O6H)-] were divided by the peak areas arising from the internal standard transition m/z 426 [(dGDP - H)-] f 159 [(P2O6H)-]. The relative increase in the ratio was expressed as a function of time for each individual inhibitor concentration held at fixed levels of substrate. The slope of the line generated from each inhibitor concentration was divided by the slope of the standard curve to obtain a quantitative value of the enzyme velocity expressed in units of concentration of GDP per minute. Kinetic Analysis. The Ki for each inhibitor was determined using nonlinear regression of the data fit to the equation for competitive inhibition (eq 125). Fitting of the data was performed

sion was performed in order to display the mode of inhibition via Dixon analysis using the program GraphPad Prism. The regression analysis was weighted toward the data with the lowest inhibitor concentration. Chemical Stability of 1 in the Presence of Fuc-T V. Reactions were done in tubes containing 20 mM Bis-Tris (pH 6.8), 10 mM MnCl2, 2 mM DTT, 24 mM LacNAc, and 0.3 mM 1 in 95.5 µL. The reactions were started by the addition of 0.45 munit of Fuc-T V (4.5 µL of the diluted enzyme preparation) and incubated at 37 °C for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h. For the control reaction, 4.5 µL of water was added in place of the enzyme and treated identically. Reactions were terminated by addition of 5 µL of the reaction mixture into a tube containing 30 µL of 70% MeOH and 0.5 nmol of dGDP as an internal standard. The samples were then analyzed by ESI-MS/MRM as described above by monitoring the inhibitor transition m/z 584 [(1 - H)-] f 301 [(C7H11O9P2)-], a predicted enzymatically generated metabolite of 1 m/z 442 [(GDP - H)-] f 159 [(P2O6H)-], and the internal standard transition m/z 426 [(dGDP - H)-] f 159 [(P2O6H)-]. To determine whether the decrease in 1 was statistically significant, the least-squares fitted line through the data was tested for a slope that differed significantly from the control (F-test) with p set to g0.05.

v ) Vmax[GDP-Fuc]/{Km (1 + [I]/Ki) + [GDP-Fuc]} (1)

RESULTS AND DISCUSSION The negative ion ESI-MS spectrum, of 1 revealed a base peak corresponding to the singly charged (M - H)- ion at m/z 583.9 (calcd 584.1 Da) (Figure 1 A). The negative ion MS/MS spectrum of 1 contained structurally diagnostic fragment ions suitable for

using the GraphPad Prism program (San Diego, CA) with both Vmax and Ki set as variables. Each Ki value was calculated as the average from the independent curves arising from different constant concentrations of the substrate GDP-Fuc. Linear regres6026

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(25) Motulsky, H. J.; Ransnas, L. A. FASEB J. 1987, 1, 365-374.

Figure 2. MRM profile representing the ability to monitor multiple species during the inhibiton reaction (Dixon analysis) with 1 as the inhibitor. GDP, dGDP, GDP-Fuc, and 1 were monitored simultaneously for each injection. The specific transitions were generated by collisionally activated dissociation of the respective parent ions. Row 1: GDP [m/z 442 (GDP - H)- f 159 (P2O6H)-]. Row 2: dGDP [m/z 426 (dGDP - H)- f 159 (P2O6H)-]. Row 3: GDP-Fuc [m/z 588 (GDP - Fuc-H)- f 442 (GDP - H)-]. Row 4: 1 [m/z 584 (1 - H)f 301 [(C7H11O9P2)-]. For the data represented here, the concentrations of 1 were (A) 0.00, (B) 0.02, (C) 0.05, (D) 0.10, (E) 0.20, and (F) 0.30 mM, respectively, at a fixed concentration of GDP-Fuc (0.05 mM). For each concentration of 1, the reaction was analyzed at four different time points (0, 6, 12, and 18 min, from left to right within each bracket). For the kinetic data, the transition of GDP (row 1) was monitored with reference to the transition of the internal standard dGDP (row 2).

MRM analysis: (PO3)- at m/z 79.4 (calcd 79.0 Da), (HO6P2)- at m/z 158.9 (calcd 158.9 Da), (C7H11O9P2)- at m/z 300.8 (calcd 300.9 Da), (C10H11N5O7P)- at m/z 344.9 (calcd 344.0 Da), (C10H13N5O8P)at m/z 361.9 (calcd 362.1 Da), (C10H12N5O10P2)- at m/z 423.8 (calcd 424.0 Da), and (C10H14N5O11P2)- at m/z 442.0 (calcd 442.0 Da) (Figure 1 B). The m/z 300.8 fragment ion was the most abundant and therefore chosen to monitor 1 during the reaction progress in subsequent experiments. A representation of the raw data from MRM analysis of a typical inhibition assay containing 1 is shown in Figure 2. The components that could be observed were LacNac (data not shown), GDP, dGDP, GDP-Fuc, and 1 or GMP. The enzyme product (GDP) and the internal standard (dGDP) were used to monitor enzyme activity as previously described.15 The dynamic changes in the concentration of each component shown in Figure 2, followed the expected trend for Dixon analysis. The Dixon analysis of the known competitive inhibitor GMP (Figure 3 A) indicated a pattern of competitive inhibition, where the intersection of the two lines defined the Ki.26 Nonlinear regression of the data fit to eq 1 for competitive inhibition gave a Ki of 211.8 ( 24.7 µM (n ) 3). To validate the data obtained by ESI-MS/MRM, the experiment was performed in identical condi(26) Dixon, M.; Webb, E. D. Enzymes; Academic Press: New York, 1964; pp 328-329.

Figure 3. (A) Dixon analysis showing a signature plot for competitive inhibition for GMP, a known inhibitor of Fuc-Ts, at the indicated concentrations of GDP-Fuc. (B) Dixon analysis showing a signature plot for competitive inhibition for 1. All Ki values were determined by nonlinear regression of the data fit to eq 1.

tions using the radioactive assay method with GDP-[U-14C]Fuc. Nonlinear regression of the data fit to eq 1 gave a Ki of 235.6 ( 59.4 µM (n ) 3), which is statistically indistinguishable from the values obtained by ESI-MS/MRM. These data clearly show that ESI-MS/MRM can be used for the kinetic characterization of inhibitors with good accuracy, reproducibility, and adequate sensitivity for reaction mixtures containing as little as 10 pmol/ µL product using flow injection analysis at 10 µL/min with a conventional electrospray source. The selectivity provided by MS/ MS reduces the likelihood of contamination of the analyte signals from ions of the same nominal m/z, which can affect the reproducibility of data acquired, for example, during selected ion monitoring (SIM).15 Considering that a relatively small error in the estimation of analyte ion intensities can translate into relatively large errors in the kinetic value estimates (Ki, Km), the use of MS/MS-based techniques provides a powerful advantage. The Ki values reported here for GMP are significantly lower than the 700 µM previously reported for this reaction16 using the standard radioactivity-based assay at pH 6.2. This value could not be reproduced by either experimental method (ESI-MS/MRM or the conventional radioactivity-based method). The influence of pH on Ki was investigated by running the inhibition assay for GMP at pH 6.2, as reported, rather than at pH 6.8. This yielded a Ki of 180 ( 18.7 µM (n ) 1), which is not statistically different from our results at pH 6.8. This stresses the importance of taking caution when kinetic values from nonidentical conditions are compared within the literature. The inhibition profile for 1 revealed by Dixon analysis (Figure 3B) is characteristic for a competitive inhibitor with Ki estimated Analytical Chemistry, Vol. 73, No. 24, December 15, 2001

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Figure 4. (A) Proposed transition state of the Fuc-T-catalyzed reaction representing the flattened oxocarbenium ion-like conformation of the fucosyl residue. (B) The structure of 1, an unsaturated carbocyclic analogue of GDP-Fuc, designed to mimic the flattened half-chair conformation of the transition state. (C) It was speculated that 1 might be both a substrate and an inhibitor or that it might form a covalent linkage with the enzyme (a suicide-type inhibitor). The upper path shows the transfer of 1 to the acceptor sugar, and the lower path shows covalent binding of 1 to a nucleophilic amino acid side chain within the enzyme active site.

at 25.6 ( 2.8 µM (n ) 3). The structure of 1 was designed to mimic the flattened anomeric conformation of the fucosyl residue (Figure 4A and B). With respect to the structure of 1, the potency of inhibition strongly agrees with a flattened half-chair conformation of the fucose residue during the transition state, which is consistent with both of the currently proposed mechanisms for Fuc-T V.17,27 Because the allylic phosphate is a good leaving group, it was originally speculated that the inhibitor 1 might act as a slow substrate for Fuc-T V or might form a covalent linkage with a nucleophile within the active site of the enzyme (Figure 4C). Because ESI-MS/MRM allows the analysis of multiple components of the enzymatic reaction, the transition from 1, m/z 584 (M - H)-, in the presence and absence of Fuc-T V was analyzed directly over time to test these hypotheses. The chemical state of 1 (m/z 584 f 301) together with the formation of a predicted metabolite of 1, GDP (m/z 442 f 159), was monitored simultaneously over 10 h in the presence and absence of Fuc-T V (Figure 5). There was no statistically significant change (p ) 0.140 > 0.05) in the intensity of the inhibitor-specific transition (m/z 584 f 301), indicating no reduction in the concentration of 1. Likewise there was no increase in the intensity of the GDP-specific transition (m/z 442 f 159), showing that there was no detectable formation of GDP. In addition, MS/MS in both positive and negative modes gave no evidence for the formation of the predicted corresponding pseudo-LeX trisaccharide product at m/z 526.2 (M + H)+ or 524.2 (M - H)-, respectively. These data clearly indicate that the inhibitor 1 is not accepted as a substrate for Fuc-T V. Thus, (27) Smith, S. L.; Compston, C. A.; Palcic, M. M.; Bamford, M. J.; Britten, C. J.; Field, R. A. Biochem. Soc. Trans. 1997, 25, 5, S630.

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Figure 5. Simultaneous monitoring of the transitions for both 1 (m/z 584 f 301) and GDP (m/z 442 f 159), a predicted metabolite of 1, in the presence of Fuc-T V via ESI-MS/MRM. There was a statistically insignificant change in the concentration of 1 and no production of GDP during the enzymatic reaction, thus indicating that 1 serves as a chemically stable inhibitor of Fuc-T V and does not act as a substrate, nor does it form any covalent linkage with the enzyme.

ESI-MS/MRM has confirmed the chemical fate of 1, allowing an extensive understanding of its mechanism of inhibition by a direct analysis of the reaction mixture during the reaction progress. This direct method is straightforward as compared to more indirect methods thus far employed.17,28,29 Thus, the ability to monitor the chemical state of inhibitors during the reaction progress, or a specific metabolite, will provide valuable insights into our understanding of enzyme-inhibitor interactions. Further application of

ESI-MS/MRM for the characterization of various synthetic inhibitors for Fuc-T V is currently being investigated. CONCLUSION The data presented here indicate that MRM will allow the acquisition of accurate kinetic values (Ki, Km) from crude reaction mixtures. As a result, ESI-MS/MRM serves as a powerful method for the kinetic characterization of inhibitors. Furthermore, ESIMS/MRM allows direct monitoring of the inhibitor during the reaction progress, which leads to a more comprehensive understanding regarding the mechanism of action of the inhibitor than existing traditional methods would allow. (28) McCarter, J. D.; Adam, M. J.; Braun, C.; Namchuk, M.; Tull, D.; Withers, S. G. Carbohydr. Res. 1993, 249, 77-90. (29) Withers, S. G.; Rupitz, K.; Street, I. P. J. Biol. Chem. 1988, 263, 7929-32.

ACKNOWLEDGMENT This paper is dedicated to Professor Kenneth L. Rinehart, our long-term mentor, collaborator, and friend, on the occasion of his retirement from University of Illinois at UrbanasChampaign. We thank Professors Jon M. Fukuto and Arthur K. Cho for advice and helpful criticism. This work was supported by the Crump Institute for Molecular Imaging and the Department of Molecular & Medical Pharmacology, UCLA School of Medicine. The W. M. Keck Foundation provided support toward instrument purchase.

Received for review July 26, 2001. Accepted October 12, 2001. AC015574G

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