Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry

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Analytical Biochemistry 341 (2005) 94–104

ANALYTICAL

BIOCHEMISTRY

Kinetic measurements and mechanism determinationof Stf0 sulfotransferase using mass spectrometry

Na Pi a,b, Mike B. Hoang b, Hong Gao a,b, Joseph D. Mougous c,d,Carolyn R. Bertozzi b,c,d, Julie A. Leary a,b,*

a Department of Chemistry and Division of Molecular and Cellular Biology, Genome Center, University of California, Davis, CA 95606, USAb Department of Chemistry, University of California, Berkeley, CA 94720, USA

c Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USAd Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA

Received 12 January 2005Available online 14 March 2005

Abstract

Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3 0-phosphoadenosine-5 0-phospho-sulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfatedmetabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ioniza-tion (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfurylgroup transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transformion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0–PAPS and Stf0–trehalose binary complexes, and a Stf0–3 0-phosphoadenosine 5 0-phosphate–trehalose ternary complex. The results from our studystrongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate.In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the firstdetailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reactionpathway and catalytic mechanism of promising enzymatic systems.� 2005 Elsevier Inc. All rights reserved.

Keywords: Stf0; Mass spectrometry; ESI-MS assay; Rapid equilibrium random sequential Bi-Bi mechanism

Sulfation of biomolecules is a posttranslational mod-ification that is widely observed from bacteria to mam-mals and regulates a variety of cellular communicationevents [1,2]. Sulfotransferases (STs)1 are enzymes thatare responsible for transferring the sulfuryl group from

0003-2697/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2005.02.004

* Corresponding author. Fax: +1 530 754 9658.E-mail address: [email protected] (J.A. Leary).

1 Abbreviations used: ST, sulfotransferase; PAPS, 30-phosphoadenosine 50-3 0-phosphate binding motif; EST, estrogen sulfotransferase; ER, estrogen recesulfate N-deacetylase/N-sulfotransferase 1; HS3OST-1, heparan sulfate 3-O-ssulfotransferase; SL-1, sulfolipid-1; T2S, trehalose-2-sulfate; ESI-MS, electdesorption/ionization; FT-ICR MS, Fourier transform ion cyclotron rephosphoglucoisomerase; PGM, phosphoglucomutase; SIM, selected ion mGalNAc-6S.

a universal sulfuryl group donor, 3 0-phosphoadenosine5 0-phosphosulfate (PAPS), to numerous acceptor sub-strates including proteins [3,4], carbohydrates [1,5],and low-molecular-weight metabolites [6,7]. They alsoplay significant roles in modulating normal and patho-

phosphosulfate; PAP, 3 0-phosphoadenosine 5 0-phosphate; 3 0PB motif,ptor; 5 0PSB-loop, 50-phosphosulfate binding loop; HNDST-1, heparanulfotransferase isoform 1; PST, phenol sulfotransferase; FST, flavonolrospray ionization mass spectrometry; MALDI, matrix-assisted lasersonance mass spectrometry; GST, glutathione S-transferase; PGI,onitoring; GalNAc, N-acetylgalactosamine; DDi-6S, a-DUA-[1fi 3]-

mailto:[email protected]

Stf0 mechanism study using mass spectrometry / N. Pi et al. / Anal. Biochem. 341 (2005) 94–104 95

genic biological processes. For example, bacterial sulfo-transferase NodH is involved in the biosynthesis of sul-fated nod factor which induces root nodulation in thesymbiotic host plant [8], while the interaction betweenL-selectin and its sulfated glycoprotein ligands contrib-utes to the leukocyte immigration into inflamed tissuesand results in acute and chronic inflammation [9,10].The sulfated chemokine receptor CCR5, modified bytyrosine protein sulfotransferases, binds to HIV gp120during the HIV infection process [11]. Similarly, the sul-fation of estradiol, catalyzed by estrogen sulfotransfer-ase (EST), can activate the estrogen receptor (ER) andis believed to underlie the genesis of breast cancer[12,13]. The discovery that sulfotransferases participatein various disease states has positioned the enzymes asnovel therapeutic targets [1,2].

The mechanistic information for sulfuryl group trans-fer is critical in understanding the functions of STs invivo. Studies of the structure and catalytic mechanismof this enzyme class have been most extensively focusedon eukaryotes, particularly on cytosolic STs. Crystallo-graphic and mutational analyses have provided criticalinformation about the active-site residues involved inST catalysis [14–22]. For example, the crystal structuresof EST have been solved in the presence of 3 0-phospho-adenosine 5 0-phosphate (PAP) and estradiol [14], PAPand vanadate [15], and mostly recently with the donorsubstrate PAPS [16]. These EST structures, along withsite-directed mutagenesis work, have unraveled the cen-tral catalytic roles for Lys and Thr in the 5 0-phosphosul-fate binding loop (5 0PSB-loop) and for Ser in the 3 0-phosphate binding motif (3 0PB motif). Structural studiesof the ST domain of the heparan sulfate N-deacetylase/N-sulfotransferase 1 (HNDST-1) [21] and the heparansulfate 3-O-sulfotransferase isoform 1 (HS3OST-1) [22]associated with PAP further verified Lys, His, and Seras highly conserved residues. The relative orientation

Scheme 1. Stf0-catalyzed sulfuryl group transfer reaction from PAPS t

of the bound molecules in the solved ST structures indi-cated a characteristic transition state for a SN2-like in-line displacement reaction [16,22,23]. In addition, directkinetic analyses have been performed to investigate thecatalytic mechanism of cytosolic STs. Varin and co-workers [24,25] determined that phenol sulfotransferases(PSTs) and flavonol sulfotransferases (FSTs) followedan ordered Bi-Bi mechanism using initial rate kineticsand product inhibition. Recently, a random Bi-Bi mech-anism was elucidated for EST [26] and an insect sulfo-transferase, retinol dehydratase [27]. The kineticstudies point toward a sequential mechanism with ter-nary complex formation for cytosolic STs, and this find-ing is consistent with a previously proposed in-linetransfer mechanism based on the structural features ofcytosolic STs.

In comparison, only sparse information on the struc-ture and mechanism of bacterial STs has been obtained.Only one bacterial ST, NodH, from Rhizobium meliloti,has been assigned a hybrid random ping-pong mecha-nism using kinetic analysis and characterization of thesulfated enzyme intermediate [28]. In this mechanism,both donor and acceptor substrates bind independentlyand randomly at two different sites on NodH. A flexibledomain which presumably contains the 5 0PSB-loop ofNodH is sulfated by PAPS bound in the first site, yield-ing the substituted enzyme intermediate.

Recently, a novel mycobacterial sulfotransferase,termed Stf0, was identified and characterized by Mou-gous et al. [29]. This enzyme initiates the biosynthesisof sulfolipid-1 (SL-1), the most abundant sulfatedmetabolite and potential virulence factor found inMycobacterium tuberculosis [30–33]. Stf0 was demon-strated to catalyze the sulfuryl group transfer fromPAPS to trehalose to form trehalose-2-sulfate (T2S),the core disaccharide of SL-1 (Scheme 1). The crystalstructure of Stf0 was solved in the presence of trehalose,

o trehalose, generating PAP and trehalose-2-sulfate as products.

96 Stf0 mechanism study using mass spectrometry / N. Pi et al. / Anal. Biochem. 341 (2005) 94–104

which revealed the molecular basis of trehalose recogni-tion. As the first prokaryotic ST to be structurally char-acterized and the first carbohydrate ST that has itsstructure solved in the presence of an acceptor substrate,Stf0 is an interesting target for catalytic mechanismdetermination.

Soft ionization methods such as electrospray ioniza-tion (ESI) and matrix-assisted laser desorption/ioniza-tion mass spectrometry have been demonstrated to betechniques complementary to conventional spectropho-tometric methods for enzyme kinetics studies [34–42].A facile and broadly applicable ESI-MS assay devel-oped by Leary and co-workers has been implementedfor kinetic analyses and substrate specificity evaluationof several enzyme systems such as glutathione S-trans-ferase [36], hexokinase [37], PGI and PGM [38,39],and NodH sulfotransferase [28,40,41]. This assay doesnot require chromophogenic reaction components andthe total analysis time is comparable to that of tradi-tional methods. Additionally, assays for analyzing mul-tiple substrates [41] and multiple enzyme tandemreactions were developed using the ESI-MS techniqueand the latter were successfully applied to monitor thebiosynthetic pathway of the antibiotic novobiocin [42].

Herein we report the complete kinetic characteriza-tion of the Stf0-catalyzed sulfuryl group transferreaction from PAPS to trehalose. The product, treha-lose-2-sulfate, was quantified in relation to an internalstandard using single-point normalization factors. Theenzyme kinetic parameters for Stf0 were rapidlymeasured using the ESI-MS assay. The catalytic mecha-nism of Stf0 was determined by initial rate kineticsanalysis and product inhibition study and confirmedby identification of the noncovalent ternary complexintermediate using FT-ICR mass spectrometry. Theseare the first data reported on the catalytic mechanismof mycobacterial carbohydrate sulfotransferase Stf0and the data are highly suggestive of a rapid equilibriumrandom sequential Bi-Bi mechanism. Our research onthe enzymatic properties of Stf0 provides a platformfor understanding the roles of STs in bacteria. Consider-ing that prokaryotic carbohydrate STs (e.g., NodH andStf0) are functionally analogous to eukaryotic carbohy-drate STs that have a central role in numerous diseasestates, insights gained from the structure and mechanismstudy of Stf0 will greatly facilitate ST-targeted drugdesign [43].

Materials and methods

General materials and methods

Trehalose, 3 0-phosphoadenosine 5 0-phosphate, andthe internal standard a-DUA-[1 fi 3]-GalNAc-6S (DDi-6S) were purchased from Sigma (St. Louis, MO) and

used without further purification, while 3 0-phosphoade-nosine-5 0-phosphosulfate was purchased from Calbio-chem (San Diego, CA). Stf0 was overexpressed andpurified as previously described [29]. Coomassie plusprotein assay reagent (950 mL) and albumin standard(10 · 1 mL) were purchased from Pierce (Rockford,IL). All enzymatic reactions and mass spectrometricanalyses were carried out at ambient temperature.

PAPS purification

Given that commercial PAPS was found to contain15% PAP, a product inhibitor of the Stf0 enzyme,further purification of PAPS was performed to ensureaccurate results for Stf0 kinetics. PAPS was purified viaa 1-mL HiTrap Q HP anion-exchange column (Amer-sham, Piscataway, NJ) using ddH2O and 2 M NH4OAc,pH 6.8, in a mixture gradient of 2–100% 2 M NH4OAcover 90 min at constant 4 �C. A BioLogic LP system(Bio-Rad, Hercules, CA) was used to perform the chro-matography. Aliquots of 700 lL were automatically col-lected every 90 s. The presence of PAPS and PAP in theeluate was monitored using UV absorption at 254 nm.Fractions containing separated PAPS were collectedand subsequently dialyzed in ddH2O using 3-lL-capacitydialysis tubes with 100 MW cutoff (Spectrum Lab, Ran-cho Dominguez, CA) at 4 �C. The total concentrationof PAPS after desalination was determined by UV at260 nm (e = 15,100 M�1 cm�1). The purity of PAPS afterdialysis was measured to be 90% usingmass spectrometryas previously described [44].

Instrumentation

Ion trap mass spectrometry

A LCQ classic ion trap mass spectrometer equippedwith an ESI source and a HPLC pump (Thermo-Finni-gan, San Jose, CA) was used for kinetic analyses.Approximately 10 lL of each sample solution was in-jected into a 5-lL injection loop and delivered via anLC pump using a mixture of water and methanol at aflow rate of 20 lL min�1. The capillary temperatureand the spray voltage were kept at 200 �C and 3.2 kV,respectively. The ions of interest were monitored in thenegative ion mode and signal intensity was optimizedusing the automatic tuning option on the instrument.When the signal intensity for one sample decreased fromapproximately 5 · 105 detector counts per scan to5 · 103 detector counts per scan, indicating the con-sumption of the sample, the next sample was injected.Selected ion monitoring (SIM) mode was employed forquantification and the peak area for each SIM ion wasautomatically integrated using the Qual Browser pro-gram. The integrated peak area of the product ion andthe internal standard ion were used to determine theirpeak area ratio (AP/AIS).


FT-ICR mass spectrometry

A Bruker FT-ICR mass spectrometer equipped withan actively shielded 7-Tesla superconducting magnetwas used for analyzing the Stf0–ligand noncovalentcomplexes. Solutions were infused into an Apollo elec-trospray source at a rate of 2 lL min�1. The N2 nebuliz-ing and drying gas pressures were maintained at 50 and25 psi, respectively. The bias on the glass capillary waskept at 4600 V and a 182 �C drying gas was used to as-sist the desolvation process. Further desolvation wasachieved by collisions of the ions with neutral buffergas at the nozzle–skimmer region using a �150-V capil-lary exit accelerating voltage. A throttle valve was in-stalled at the nozzle–skimmer region and the pressurewas adjusted to 1 · 10�5 mbar. Ions were externallyaccumulated in a radiofrequency-only hexapole for 1 sand four ion packages were transferred into the ICRcell. Excessive kinetic energy was removed by collidingthe ions with Ar pulsed into the cell to a pressure of�10�8 mbar and the trapping voltage was decreased to�0.5 V. A series of pump downs were applied to lowerthe pressure in the cell to �10�10 mbar for ion detection,which was achieved by chirp excitation and broad-banddata acquisition using an average of 16 time domaintransients containing 8 k data points. The original timedomain free induction decay spectra were zero-filled,Gaussian-multiplied, and Fourier-transformed. Theparameters of the ESI source, ion optics, and cell weretuned for the best signal to noise ratio and kept forthe same set of experiments. All the data were acquiredand processed using Bruker Xmass v6.0.0 software(Billerica, MA).

Enzyme kinetics

Quantification method

Single-point normalization factors (R factors) [37,40]were used for product quantification. As described inEq. (1), the peak area integration ratio of the production and the internal standard ion (AP/AIS) is related totheir concentration ratio through an R factor.

R ¼ ðAP=AISÞ=ð½Product�=½Internal standard�Þ: ð1ÞIn our kinetic study, a chondroitin disaccharide, a-DUA-[1fi 3]-GalNAc-6S (DDi-6S), was used as aninternal standard because of its similar molecular weightand chemical structure relative to the product, T2S. Thetwo ions monitored were [DDi-6S]

� and [T2S]�, at m/z421 and m/z 503, respectively.

To obtain R factors at different trehalose concentra-tions, nine samples containing nine trehalose concentra-tions (0, 2, 4, 6, 8, 10, 15, 20, 30 mM) and a knownproduct and internal standard concentration (50 lM)were prepared. SIM was performed in triplicate on eachsample to obtain AP/AIS, and R factors at different tre-halose concentrations were calculated using Eq. (1).

For each quenched reaction sample analyzed, theproduct concentration can be calculated via Eq. (2)using the ESI-MS data (AP/AIS) and the correspondingR factor determined at the trehalose concentration usedfor the reaction.

½Product� ¼ ðAP=AISÞ � ½Internal Standard�=R: ð2Þ

Kinetic constant measurements

All reactions were assayed in 30-lL 10 mM NH4OAcbuffer, pH 7.5. Reactions were initiated by the additionof Stf0 and terminated at specified time points byquenching 10-lL aliquots in 40 lL of methanol contain-ing 12.5 lM DDi-6S at 4 �C. The quenched samples weresubsequently analyzed by ESI-MS. For the determina-tion of kinetic parameters for trehalose, Stf0 and PAPSwere maintained at 112 nM and 1 mM, respectively, andtrehalose was varied over a concentration range of 2–30 mM. For the determination of kinetic parametersfor PAPS, the concentrations used for Stf0 and trehalosewere 112 nM and 30 mM, respectively, and PAPS con-centration was varied from 10 to 500 lM. Both satura-tion and double-reciprocal plots were generated vianonlinear fit to Michaelis–Menten equation using theGraFit program (Version 4.0.12; Erithacus Software,Horley, Surrey, UK). The Km and kcat values for treha-lose and PAPS were subsequently determined using thesame program. Each experiment was carried out intriplicate.

Initial rate kinetics analysis

The mechanism of Stf0 was determined using treha-lose and PAPS as substrates. Stf0 was added to a finalconcentration of 250 nM. The concentration of PAPSwas varied from 10 to 50 lM, and the concentrationof trehalose ranged from 2 to 10 mM. Partial substrateinhibition by trehalose and PAPS is negligible at thisconcentration range. Initial velocities were determinedunder each of the 25 conditions defined by a 5 · 5 matrixof substrate concentrations. The catalytic mechanism ofStf0 was evaluated by plotting 1/V0 as a function of both1/[PAPS] at different fixed trehalose concentrations and1/[trehalose] at different fixed PAPS concentrationsusing the GraFit program. Experiments were carriedout in triplicate.

Product inhibition study

For the product inhibition study of PAP versusPAPS, Stf0 was added to a final concentration of100 nM, while trehalose was kept at a constant concen-tration of 10 mM. The concentration of PAPS wasvaried from 10 to 50 lM, and the five different concen-trations used for PAP were 0, 10, 20, 50, and 100 lM.For the product inhibition study of PAP versus treha-lose, the concentrations used for Stf0 and PAPS were100 nM and 200 lM, respectively. The concentration


of trehalose was varied from 2 to 10 mM, and the fivePAP concentrations used were 0, 50, 100, 150, and200 lM. In each case, the 25 quenched samples wereanalyzed by ESI-MS to obtain initial velocities. Themode of inhibition and the Ki value were determinedby fitting data into the GraFit program and displayedas a double-reciprocal plot. Each experiment was carriedout in triplicate.

Detection of the Stf0 noncovalent complexes

A series of five solutions were prepared in 50 mMNH4OAc, pH 7.5. Solution 1 contained 10 lM Stf0;solution 2 contained 100 lM trehalose and 10 lM Stf0;solution 3 contained 20 lM PAP and 10 lM Stf0; solu-tion 4 contained 20 lM PAPS and 10 lM Stf0; solution5 contained 100 lM trehalose, 20 lM PAP, and 10 lMStf0. Each solution was previously incubated on ice for20 min and then infused into the ESI-FT-ICRmass spec-trometer using positive ion mode detection. The spraychamber was wrapped with ice bags to prevent the pro-tein sample from precipitating out from the buffersolution.

Results and discussion

Product quantification

The trehalose concentrations used in the Stf0 kineticanalysis were in the range of 2–30 mM which was muchhigher than the concentrations of the other reactioncomponents (PAPS, PAP, and T2S), including the inter-nal standard. Trehalose in this concentration range willaffect the relative ionization intensity ratio of the prod-uct and the internal standard, therefore affecting the R

factor. To achieve accurate product quantification, R

factors were measured at different trehalose concentra-tions and subsequently used in the kinetic analysis.

Fig. 1. Product quantification was carried out using different normal-ization factors (R factors) at different trehalose concentrations. Shownhere is the plot of R factor versus [trehalose].

As illustrated in Fig. 1, the average R factor was deter-mined to be 0.6 when no trehalose was added to thereaction solution, and it increased gradually over thetrehalose concentration range of 0–15 mM. The highestR factor was observed at a trehalose concentration of15 mM. Over the trehalose concentration range of 15to 30 mM, the R factor was in a relatively steady state.This is likely due to a higher ionization suppression bytrehalose on the internal standard ion than on the prod-uct ion, resulting in the increased R factor with increasedtrehalose concentration.

Kinetic constant measurements

The kinetic parameters of Stf0 were defined witheither PAPS or trehalose as the variable substrate (Figs.2A and B). The results were obtained by fitting data intothe Michaelis–Menten equation. No enzyme inhibitionwas observed over the concentration ranges of PAPSand trehalose used. Typical hyperbolic saturationwas observed when PAPS was the variable substrate

Fig. 2. Measurements of kinetic constants for Stf0-catalyzed sulfurylgroup transfer reaction from PAPS to trehalose. (A) Saturation plot ofV0 versus [PAPS] and double-reciprocal plot of 1/V0 versus 1/[PAPS](inset) at a fixed trehalose concentration (30 mM). (B) Saturationplot of V0 versus [trehalose] and double-reciprocal plot of 1/V0 versus1/[trehalose] (inset) at a fixed PAPS concentration (1 mM).

Fig. 3. Initial rate kinetics analysis for mechanism determination ofStf0-catalyzed sulfuryl group transfer reaction. Double-reciprocalplots of 1/V0 versus 1/[PAPS] at different fixed trehalose concentra-tions. (s [trehalose] = 2 mM, d [trehalose] = 4 mM, h [treha-lose] = 6 mM, j [trehalose] = 8 mM, and n [trehalose] = 10 mM).


(trehalose concentration was held constant at 30 mM),resulting in an apparent Km of 18 ± 3 lM for PAPS.Under these conditions, a maximum rate constant of96 ± 2 min�1 was observed and this is in good agree-ment with the rate constant measured in parallel exper-iments carried out using 30 mM trehalose undersaturating PAPS conditions (98 ± 2 min�1). When tre-halose was the variable substrate and PAPS concentra-tion was held constant at 1 mM, a Michaelis–Mentencurve fit of the data up to 30 mM trehalose providedan apparent Km of 15 ± 1 mM and a kcat of134 ± 10 min�1 for trehalose. The kinetic constants fortrehalose are in excellent agreement with those obtainedby the TLC assay used previously [29]. The results forPAPS are the first reported values to date and the Km va-lue of PAPS is similar to those measured for other STs[24–27,40]. The Km values of trehalose and PAPS indi-cate that the donor substrate PAPS has a much higherbinding affinity to Stf0 than the acceptor substrate tre-halose. This is reasonable given that trehalose is presentat high physiological concentrations in vivo [45], sug-gesting that the enzyme is optimized to function at phys-iological levels of trehalose.

Initial rate kinetics analysis

To examine whether a sequential or a ping-pong Bi-Bi mechanism is operative in Stf0, a classical initialvelocity study of the sulfuryl group transfer reactionwas performed. Initial reaction rates were obtainedusing data at low reaction conversion to avoid compli-cated reaction conditions such as substrate depletion,product inhibition, and reverse reaction. Preliminaryexperiments were performed to ensure that inhibitionof both substrates was negligible within the chosen con-centration ranges. The initial rate of the reaction wasdetermined as a function PAPS at different fixed concen-trations of trehalose (Fig. 3) and as a function of treha-lose at different fixed concentrations of PAPS (data notshown). The kinetic data were fitted into the rate equa-tion of two mechanistic models (sequential and ping-pong mechanisms) of bisubstrate reactions using theGraFit program, and the best fit was obtained in thecase of a sequential mechanism model. Fig. 3 showsthe double-reciprocal plot of 1/V0 versus 1/[PAPS] andthe five lines representing different trehalose concentra-tions intersect at one point. In the sequential mecha-nism, Stf0 binds to trehalose and PAPS to form aternary complex, catalyzes the sulfuryl group transferbetween the two substrates inside the complex, and sub-sequently releases the reaction products T2S and PAP.

Product inhibition study using PAP

We tested the inhibition effect of PAP on the sulfo-transferase activity of Stf0 to obtain more information

about the catalytic mechanism. The inhibition resultswere obtained by fitting data into the rate equation ofthree inhibition patterns (competitive, noncompetitive,and mixed-type inhibitions) using the GraFit program.PAP was found to be a good inhibitor to the donor sub-strate PAPS when trehalose concentration was kept con-stant at 10 mM. The best data fit was obtained in thecase of a competitive inhibition model. The double-reci-procal plot of 1/V0 versus 1/[PAPS] at different fixedPAP concentrations (Fig. 4A) results in a family of fivelines intersecting at the y axis, unambiguously indicatingthat PAP is a competitive inhibitor of PAPS. The appar-ent Ki value for PAP was determined to be 18 ± 2 lM,which is very similar to the apparent Km value measuredfor PAPS. This result indicates that PAP and PAPS bearbinding affinities comparable to those of the target en-zyme. With regard to the acceptor substrate trehalose,PAP was found to be a very weak inhibitor under satu-rating PAPS conditions ([PAPS] = 200 lM). No detect-able product inhibition of PAP versus trehalose wasobserved when <50 lM PAP was used in the prelimin-ary experiments. When a higher PAP concentrationrange (0–200 lM) was used, weak inhibition of trehalosebinding was observed. The best data fit was obtained inthe case of a noncompetitive inhibition model. Asdepicted in Fig. 4B, the five lines representing five differ-ent PAP concentrations in the double-reciprocal plotshare a common x intercept, suggesting a noncompeti-tive inhibition mode of PAP with respect to trehalose.

Fig. 4. Product inhibition study of Stf0 by PAP. (A) Double-reciprocal plots of 1/V0 versus 1/[PAPS] at different fixed PAPconcentrations. Trehalose concentration was fixed at 10 mM. (s[PAP] = 0 lM, d [PAP] = 10 lM, h [PAP] = 20 lM, j [PAP] =50 lM, and n [PAP] = 100 lM). (B) Double-reciprocal plots of 1/V0

versus 1/[trehalose] at different fixed PAPS concentrations. PAPSconcentrations was fixed at 200 lM. (s [PAP] = 0 lM, d [PAP] =20 lM, h [PAP] = 50 lM, j [PAP] = 100 lM, and n [PAP] =200 lM).


The apparent inhibition constant, Ki, was determined tobe 405 ± 26 lM. These data taken together support asequential Bi-Bi mechanism for Stf0. There are two dis-tinctive binding sites on Stf0 for PAPS and trehalose;PAP and PAPS share and compete for one of the

binding sites, while T2S and trehalose share and com-pete for the other binding site.

Detection of Stf0-associated noncovalent complexes

An important feature of a sequential Bi-Bi mecha-nism is the formation of a ternary complex, so-called be-cause it contains the enzyme and both substrates in asingle complex. Detection of the ternary complex inter-mediate of a transferase enzyme and its substrates ishighly supportive of a sequential catalytic mechanismadopted by the enzyme [46]. Unfortunately, most grouptransfer reactions have a relatively high reaction turn-over number, and the enzyme–substrates ternary com-plex is rapidly converted to the free enzyme and thereaction products. Therefore, the ternary complex maynot exist in the reaction solution long enough to be de-tected. This explanation is probably the reason that theStf0–PAPS–trehalose complex could not be detectedwhen Stf0 was incubated with PAPS and trehalose.However, a ternary complex between Stf0, PAP, andtrehalose will accumulate in solution given that Stf0 fol-lows a sequential Bi-Bi mechanism and PAP binds to thePAPS binding site on Stf0. This Stf0–PAP–trehalosecomplex is analogous to the Stf0–PAPS–trehalose com-plex formed in the authentic reaction.

To verify the sequential mechanism of Stf0 catalysis,the enzyme was incubated with both PAP and trehalosein NH4OAc buffer. For comparison, Stf0 was also incu-bated with PAP, PAPS, and trehalose, individually. As acontrol, Stf0 (10 lM) was first sprayed from NH4OAcbuffer. As shown in Fig. 5A, several charge states(10+, 11+, and 12+) were observed. No dimer or highermultimeric states were observed in the mass spectrum,which suggests that Stf0 exists as a monomer in solution.When 10 lM Stf0 was incubated with 20 lM PAP, amass shift was observed at m/z 2938 in the mass spec-trum (Fig. 5B). The mass difference between[Stf0 + PAP]11+ and [Stf0]11+ was calculated to be427 Da, which corresponds to the noncovalent adduc-tion PAP. The mass spectra of 10 lM Stf0 incubatedwith 20 lM PAPS (Fig. 5C) also shifted to m/z 2945, amass difference of 507 Da indicating the formation ofStf0–PAPS binary complex. Fig. 5D represents the massspectrum of 10 lM Stf0 incubated with 100 lM treha-lose. A fivefold excess of trehalose was used because ofits relatively low binding affinity to Stf0 in solution.Compared to the spectrum of Stf0, an additional ionwas observed at m/z 2930, which was determined to be[Stf0 + trehalose]11+ based on the mass difference of341 between it and [Stf0]11+. This also clearly indicatesthe formation of a Stf0–trehalose noncovalent complex.Finally, 10 lM Stf0 was incubated with 20 lM PAP and100 lM trehalose to identify the Stf0–PAP–trehaloseternary complex (Fig. 5E). In addition to the two binarycomplex ions, [Stf0 + PAP]11+ at m/z 2938 and

Fig. 5. Detection of Stf0-associated noncovalent complexes using FT-ICR mass spectrometry. (A) Mass spectrum of 10 lM Stf0 in 50 mMNH4OAc, pH 7.5. (B) Mass spectrum of 10 lM Stf0 with 20 lM PAP in 50 mM NH4OAc, pH 7.5. (C) Mass spectrum of 10 lM Stf0 with 20 lMPAPS (90%) in 50 mM NH4OAc, pH 7.5. (D) Mass spectrum of 10 lM Stf0 with 100 lM trehalose in 50 mM NH4OAc, pH 7.5. (E) Mass spectrumof 10 lM Stf0 with 20 lM PAP and 100 lM trehalose in 50 mM NH4OAc, pH 7.5.


[Stf0 + trehalose]11+ at m/z 2930, another ion was ob-served at m/z 2969. The mass difference between thision and the [Stf0]11+ was calculated to be 768 Da, whichcorresponds to the total molecular weight of PAP andtrehalose. Thus, the noncovalent cluster was clearly ad-dressed to be [Stf0 + PAP + trehalose]11+. This resultstrongly supports the formation of Stf0–PAP–trehaloseternary complex and hence the sequential Bi-Bi catalyticmechanism of Stf0. As depicted in the spectra, there wasno charge redistribution for Stf0 upon binding to thesesubstrates or product, which suggests that the ligandsmay bind to Stf0 as the neutral forms [44]. As demon-strated in Figs. 5C and D, PAPS was able to bind toStf0 without the existence of trehalose and vice versa.These data are more supportive of a random bindingmode of substrates to the enzyme instead of a compulso-ry-order binding mode, pointing to a random sequentialBi-Bi mechanism of Stf0.

Mechanism of Stf0 catalysis

Sequential Bi-Bi and ping-pong Bi-Bi are the mostcommonly occurring catalytic mechanisms for sulfo-transferase reactions. In a sequential Bi-Bi mechanism,

both substrates bind to the enzyme to form a ternarycomplex before sulfuryl group transfer and product re-lease occurs. Sequential mechanisms can be further clas-sified as ordered sequential or random sequentialmechanisms depending on whether there is a compul-sory order for the binding of the donor and acceptorsubstrates and the release of their products. A ping-pongmechanism involves a covalently substituted enzymeintermediate, in which the sulfuryl group is transferredin a double-displacement pathway and a product is re-leased between substrate additions.

Our data in the initial rate kinetics study providestrong evidence for a sequential Bi-Bi catalytic mecha-nism for Stf0. The product inhibition patterns of PAPobtained are also consistent with a ternary complexmechanism. However, neither the data generated fromthe initial velocity analysis nor that from the inhibitionstudy of Stf0 by PAP could differentiate between an or-dered and a random mechanism. At this point, the twopossible sequential mechanisms for Stf0 could be a rapidequilibrium random sequential Bi-Bi mechanism or asteady state ordered sequential Bi-Bi mechanism. A ra-pid equilibrium ordered sequential Bi-Bi mechanism isruled out by the fact that the lines in the 1/V0 versus

Scheme 2. Rapid equilibrium random sequential Bi-Bi mechanism for Stf0-catalyzed sulfuryl group transfer reaction.


1/[PAPS] plots (Fig. 3) and the 1/V0 versus 1/[trehalose]plots (data not shown) do not intersect on the 1/V0 axis[26,46].

The identification of Stf0-associated noncovalentcomplexes further substantiates the sequential Bi-Bimechanism determined for Stf0 in the kinetic analysis.In addition, these data make it possible to arrive at areasonable hypothesis concerning the binding mode ofPAPS and trehalose to Stf0. Detection of the Stf0–PAP–trehalose ternary complex confirmed two indepen-dent binding sites on Stf0 for the donor and acceptorsubstrates. Given the facts that the Stf0–PAPS binarycomplex was formed in the absence of trehalose andthe Stf0–trehalose binary complex was formed in the ab-sence of PAPS, a random, not ordered, manner of sub-strate binding and product release is clearly morefavored. These findings suggest that a rapid equilibriumrandom sequential Bi-Bi mechanism is adopted by Stf0.As illustrated in Scheme 2, trehalose binds to both Stf0and Stf0–PAPS binary complex, and PAPS binds toboth Stf0 and Stf0–trehalose binary complex. The sulfu-ryl group is transferred from PAPS to trehalose insidethe Stf0–PAPS–trehalose ternary complex, resulting inthe formation of Stf0–PAP–T2S ternary complex. Thetwo products are subsequently released from the enzymeactive sites in a random mode, and the free enzyme, Stf0,is regenerated and continues to participate in the cata-lytic cycle. It is noteworthy that the intersection of thelines in Fig. 3 is on the horizontal axis (1/[PAPS]), whichmeans that the binding of one substrate does not changethe dissociation constant (Kd or Km) for the other sub-strate in the rapid equilibrium random sequential Bi-Bimechanism of Stf0.

Our findings on the catalytic mechanism of Stf0 areconsistent with the recently solved X-ray crystal struc-ture of Stf0 in the presence of trehalose [29]. As the firstbacterial carbohydrate ST to be structurally character-ized, Stf0 was found to share the most structural homol-ogy and conserved catalytic residues with Golgi-residentcarbohydrate STs, including HNDST-1 and HS3OST-1[21,22]. Based on the superposition of Stf0–trehalosecomplex structure with the structure of EST solved inthe presence of PAPS and estradiol, an SN2-like in-linedisplacement mechanism was postulated for Stf0 [29].Our work further addresses the mechanistic details ofStf0, and the ternary complex formed in the proposedrapid equilibrium random sequential Bi-Bi mechanism

for Stf0 supports the associative transition state in thein-line sulfuryl transfer mechanism as revealed by thestructure.

Conclusions

Initial rate kinetics analyses, product inhibition study,and experiments in noncovalent complex identificationhave been used to delineate the catalytic mechanism ofthe bacterial carbohydrate sulfotransferase, Stf0. Rapidkinetic analyses of Stf0 were performed using an electro-spray ionization mass spectrometry assay. This assay isparticularly useful in characterizing enzymes for whichno spectrophotometric assay is feasible. In addition tothe compelling kinetic data, Stf0–PAPS and Stf0–treha-lose binary complexes and Stf0–PAP–trehalose ternarycomplex were identified by FT-ICR mass spectrometry.Given both the kinetic and the noncovalent complexdata, a rapid equilibrium random sequential Bi-Bi mech-anism is evident for the catalysis of Stf0, and this result isconsistent with the Stf0 structure data. The amino acidsequence in the catalytic active sites of Stf0 has been com-pared to that of cytosolic STs, Golgi-resident carbohy-drate STs, and another bacterial carbohydrate ST,NodH. The conserved Arg15, Ser152, andGlu136 in the ac-tive sites of Stf0 are essential residues for the catalyticfunctions of Stf0, and the Ser is proposed to play a signif-icant role in regulating the sequential mechanism. Ourpresent study provides valuable information toward acomprehensive understanding of the structure, mecha-nism, and function of biologically important sul-fotransferases using mass spectrometry.

Acknowledgment

The authors gratefully acknowledge the NationalInstitute of Health (NIH), GM63521, for financialsupport.

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Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry

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