reversible inhibition of escherichia coli inorganic pyrophosphatase by fluoride: trapped catalytic...

doi:10.1016/j.jmb.2006.11.082 J. Mol. Biol. (2007) 366, 1305–1317

Reversible Inhibition of Escherichia coli InorganicPyrophosphatase by Fluoride: Trapped CatalyticIntermediates in Cryo-crystallographic Studies

V. R. Samygina,1⁎ V. M. Moiseev,2 E. V. Rodina,2 N. N. Vorobyeva2

A. N. Popov,1,3 S. A. Kurilova,2 T. I. Nazarova,2 S. M. Avaeva2

and H. D. Bartunik4

1A.V. Shubnikov Institute ofCrystallography, RussianAcademy of Sciences, LeninskyPr. 59, 119333 Moscow, Russia2A.N. Belozersky Institute ofPhysico-Chemical Biology,Moscow State UniversityLeninskie Gory, 119992Moscow, Russia3European Molecular BiologyLaboratory (EMBL), HamburgOutstation, c/o DESYNotkestrasse 85 22603Hamburg, Germany4Max Planck Unit forStructural Molecular Biology,MPG-ASMB c/o DESY,Notkestrasse 85, D-22603Hamburg, Germany

Abbreviations used: PPase, inorgaE-PPase, Escherichia coli PPase.E-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2006 E

Here, we describe high-resolution X-ray structures of Escherichia coliinorganic pyrophosphatase (E-PPase) complexed with the substrate,magnesium, or manganese pyrophosphate. The structures correspond tosteps in the catalytic synthesis of enzyme-bound pyrophosphate (PPi) in thepresence of fluoride as an inhibitor of hydrolysis. The catalytic reactionintermediates were trapped applying a new method that we developed forinitiating hydrolytic activity in the E-PPase crystal. X-ray structures wereobtained for three consecutive states of the enzyme in the course ofhydrolysis. Comparative analysis of these structures showed that the Mn2+-supported hydrolysis of the phosphoanhydride bond is followed by a fastrelease of the leaving phosphate from the P1 site. The electrophilic phosphateP2 is trapped in the “down” conformation. Its movement into the “up”position most likely represents the rate-limiting step of Mn2+-supportedhydrolysis. We further determined the crystal structure of the Arg43Glnmutant variant of E-PPase complexedwith one phosphate and fourMn ions.

© 2006 Elsevier Ltd. All rights reserved.

Keywords: pyrophosphatase; Escherichia coli; catalytic intermediates; fluor-ide inhibition; catalytic mechanism
*Corresponding author
Introduction

Fluoride is widely used as an antimicrobial agentaffecting the metabolism of many pathogenicbacteria and intracellular parasites like, e.g. Strepto-coccus, Trypanosoma, and Ascaris. In millimolarconcentrations, F− inhibits a wide variety of glyco-lytic and other enzymes and diminishes the expres-sion of stress proteins and virulence factors.1,2

Inorganic pyrophosphatase (PPase) is particularlystrongly affected by fluoride. This ubiquitousenzyme catalyzes the reversible hydrolysis/synthe-sis of inorganic pyrophosphate PPi. In solution,

nic pyrophosphatase;

ng author:

lsevier Ltd. All rights reserve

hydrolysis dominates. Since the family I solublePPases are inhibited by micromolar concentrationsof fluoride,3 studying the inhibition mechanismmayalso be of practical significance.PPi is hydrolyzed by the soluble PPases via a direct

attack of a water molecule activated through itsinteraction with two metal ions. F− was foundearlier to inhibit hydrolysis by substituting theattacking nucleophile.4 As a result, the PPi/Piequilibrium in the active site is strongly shiftedtowards synthesis, so that the stable enzyme–substrate complexes can be obtained.3,4 Here, thisproperty has been used to investigate the X-raystructures of Escherichia coli PPase complexed withPPi in the presence of two different metal activators,Mg2+ or Mn2+, at very high resolution of 1.2 Å–1.3 Å. For studying the substrate conversion processin a PPase crystal, we developed a novel methodthat involved triggering a fast increase in the

d.

mailto:[email protected]

1306 Trapped Catalytic Intermediates in E. coli PPase

catalytic activity by washing out the inhibitingfluoride and subsequent trapping of structuralintermediates by repetitive flash-cooling of thecrystal. As a result, two additional X-ray structureswere obtained from a single crystal, the firstshowing a partially hydrolyzed substrate (MnPPi/Pi–F–E-PPase, 1.20 Å resolution) and the secondshowing one of the two phosphates produced byhydrolysis (MnPi–E-PPase, 1.65 Å). In addition, thestructure of an Arg43Gln mutant of E. coli PPasecomplexed with MnPi was solved at 1.05 Å resolu-tion. A comparative analysis of these crystalstructures and complementary solution studiesprovided the basis for a detailed description of thecatalytic reaction mechanism of the family I PPases.The approach that we used for trapping inter-

mediates potentially may be applied also to studiesof the reaction mechanisms of other enzymes.

Results

Structures of E-PPase with PPi stabilized by F−

Since the PPase has a much higher affinity for PPithan for Pi, some amount of synthesized PPi may befound in the active site after incubation of PPase in asolution containing metal activator and Pi. Under theconditions used in the present work, the level ofenzyme-bound PPi was 0.15 mol/mol of enzymesubunit, while addition of 0.5 mM NaF caused anincrease in this level to 0.6 mol/mol. This propertywas used to crystallize the enzyme–PPi complex froma mixture containing PPase, metal (Mg2+ or Mn2+), Piand NaF. Enzyme–PPi crystals could be grown in thepresence of either of these metal ions, despite the factthat Mn2+ failed to support synthesis in solution.The active site of the enzyme–PPi–F

− complex inboth cases contains four metal ions, one PPi and oneF−. All ligands had 100% occupancy and werelocated in the same positions in the two structures

Figure 1. Superposition of the active sites of MgPPi–F–E-P

(Figures 1 and 2(a) for MnPPi). The conformation ofthe P1 group of PPi differs slightly in the twocomplexes, due to a slight variation in the P–O–Pangle (120° in MgPPi versus 130° in MnPPi). Allmetal ions are octahedrally coordinated. The bind-ing sites for PPi and the metal ions are summarizedin Table 1. The Mg ions in the M1 and M2 sites arelinked by a common ligand that could be identifiedas F−, since the replacement of the ion by a watermolecule as a possible alternative in the structuralmodel resulted in a strong (3.6σ) difference electrondensity peak. The positions of the F ions in the twocomplexes differ by 0.15 Å. However, the F− in bothcases is positioned in line with respect to thebridging oxygen atom Ob and at a distance that issufficiently close for attack on PPi by a watermolecule (Wa) occupying the same location whenthe fluoride is washed out.

Trapping intermediates of the PPi hydrolysis

As shown earlier,5 the tolerance of E-PPase crystalsto undergo multiple transitions from room to cryotemperature may be exploited to extend the resolu-tion limit.We used this property and the reversibilityof E-PPase inhibition by fluoride6 to develop atechnique of trapping catalytic intermediates.Subsequent to collection of a diffraction data set

from a crystal of MnPPi–F–E-PPase at 100 K, thecrystal was placed in a cryo-solution without F− forinitiating hydrolysis. Immediately after the soak, thecrystal was flash-cooled again, and a new data setwas collected. It was possible to repeat thisoperation several times with one crystal. Theprocedure was accompanied by changes in the cellparameters and the mosaicity. This caused somedegradation in the resolution limit, which wasfurther affected by the total X-ray dose. Therefore,a data collection strategy was chosen that includedreasonable limitations in the number of flash-cool-ing cycles and the resolution of the first data set.

Pase (red/gray) and MnPPi–F–E-PPase (yellow/green).

1307Trapped Catalytic Intermediates in E. coli PPase

As a result, we were able to use the same crystalfor determining two further structures, one corres-ponding to an intermediate where only a part of PPiwas hydrolyzed (Figure 2(b)) and a final structurewith one phosphate remaining after hydrolysis(Figure 2(c)).

Final MnPi–E-PPase structure

FourMn2+ and one Pi are bound in the active site ofthis complex. The phosphate is located at theelectrophilic P2 site and has a reverse configurationas compared to the P2 group of PPi (P2

down)4 so thatone of its oxygen atoms (Onu) bridges twoMn2+ in theM1 and M2 sites. This finding is consistent with theassumption that P2 is a high-affinity site, while P1 isthe site at which the first phosphate is released fromhydrolysis. The binding sites for Pi and Mn2+ in M1–M3 have 85% occupancy. Their interactions with theprotein are listed inTable 2. The coordination of Pi andthe metal ions is the same as in previous structures,except for Mn2+ in the M4 site (60% occupancy). Wefound a new location for M4, designated as M4p. Incontrast to the previously observed M4 site, whereAsp42 was the only second-sphere protein ligand forMn2+ or Mg2+, the structure of MnPi–E-PPase showsthismetal ion shifted by 1.02Å from its usual position.The metal ion is liganded via a water molecule to acarboxylic group of Asp67, which also adopts a newconformation, Asp67p.

Intermediate MnPPi/Pi–F–E-PPase structure

This structure obviously corresponds to thesituation when only part of the PPase molecules inthe crystal regained hydrolytic activity. The activesite contains four Mn2+, one F−, the remainingunhydrolyzed PPi or the electrophilic phosphate P2.The occupancies of PPi and Pi are about 45% and55%, respectively. The position of PPi is approxi-mately the same as in the MnPPi–F–E-PPasestructure, and F− is observed only in this substruc-ture. The phosphate at the P2 site has P2down

conformation as in MnPi–E-PPase. Therefore, itslocation is incompatible with that of PPi. Someprotein residues like Tyr141 and Asp67, as well asseveral water molecules and Mn2+ in the M4 site,have double conformations (Figures 2(b) and 3(b))with occupancies of 45 and 55%, respectively. Thus,they may be attributed to the two substructures ofthe active site observed within the single crystal,corresponding to either the PPi or Pi conformation.In the following, the substructure of the active sitethat each one of the double positions belongs to isdesignated by upper indexes , e.g. M4p versus M4pp.

Arg43Gln E-PPase

The mutant variant Arg43Gln of E-PPase has beencrystallized in the presence of Mn2+, Pi and NaF inthe same concentrations as the wild-type (WT)enzyme. Since the guanidine group of Arg43 is thekey ligand of phosphate P1, a removal of this group

makes synthesis of PPi virtually impossible (unpub-lished data on solution studies). Correspondingly,only the structure of Arg43Gln E-PPase complexedwith Pi bound at the P2 site could be obtained(Figure 2(d)). In general, the structure is very similarto MnPi–E-PPase. P2 phosphate is in the “down”conformation, and all four metal ions are present,the fourth being bound in the M4p position. Allligands have 100% occupancy, except M4 (75%). Theside-chains of the residues Gln43, substituting forarginine, and Asp67 were observed in the samedouble conformations as in MnPPi/Pi–F–E-PPase.

pH-dependence of fluoride inhibition

Mg or Mn-supported hydrolysis of PPi by E-PPaseis inhibited by fluoride in the different ranges of itsconcentration. The results of the present work showthat the Ki value lies in the micromolar diapason forMg-supported hydrolysis (Ki=35.8 μM at pH 7.4 and5 mM Mg2+) and in the millimolar diapason(Ki=2.8 mM at 50 μM Mn2+) for Mn-supportedhydrolysis at the same pH. For both cations, the affi-nity of the enzyme–substrate complex for F− is greatlyaffected by the pH of the medium (Figure 4(a)). In thecase ofMn2+,Ki as a function of pH has aminimum atabout pH6.5. On aDixon–Webbplot, the right branchof this dependence has a slope of 0.96, showing areduction in the affinity as a result of the protonationof a single titratable group with a pKa value of 7.3(Figure 4(b)). In the case of Mg2+, an analogousdecrease in affinity is visible in the original plot, but itsslope in Dixon–Webb coordinates is very small (<0.2;Figure 4 (c)) anddoes not allowus to calculate the pKa.From these results, however, it can definitely beconcluded that the pH transition,which is responsiblefor the change in affinity of E-PPase for fluoride, isabout two pH units higher for Mg-supportedhydrolysis as compared toMn-supported hydrolysis.In order to identify the protein group that may be

responsible for the observed pH transition, a numberof mutant variants of E-PPase (Asp65Asn, Asp67Asn,Asp70Asn, Asp102Asn and Tyr55Phe) with substitu-tions of key active site residues by non-ionizablehomologs have been tested with respect to the pHdependence of fluoride inhibition of Mg-supportedhydrolysis. None of them differed much from theWTPPase in their pH dependences. PH transitions areobserved in the range from pH 8–pH 10, and theslopes of Dixon–Webb plots have values less than 0.4(see Figure 4 (c) for Asp67Asn). The only significantdifference was observed for the Asp67Asn variant,where Ki at neutral pH decreased to a substantiallylower level as compared to the wild-type enzyme.

Discussion

Mechanistic implications of trapping intermediates

The series of structures obtained here permits usto analyze the conformational changes that


accompany the hydrolysis of PPi and the subsequentrelease of the P1 phosphate.

Hydrolysis of PPi

In general, the changes in the active site confor-mation that are associated with the hydrolysis areremarkably small. The oxygen from the attackingwater molecule Wa, whose position in the structurescontaining PPi is mimicked by F−, enters P2 as Onu.As an immediate result of hydrolysis, the phospho-rus atom of the P2 phosphate has moved by 0.55 Å(Figure 3(a)), whereas the remaining three oxygenatoms of the phosphate have kept their positions,which are fixed by interactions with the protein. Theside-chain of Asp67 shifts from the initial Asp67pp

position, where its Oδ2 atomwas interacting withWa(2.47 Å), to a new location, Asp67p, where Oδ2 is inindirect contact with Onu via a water molecule(Wat553). Double positions, which were observed

Figure 2. Electron density for the active site region of E-PPPase; (c) MnPi–E-PPase; (d) R43Q–E-PPase.

for other protein residues, could be assigned to therelease of the P1 phosphate.

Release of P1 phosphate

In the structure of MnPi–E-PPase, all four bindingsites M1–M4 are occupied while only one phosphateis bound (Figure 3(a)). The same situation, occupationof all the four metal binding sites while Pi onlypopulates P2 site,was also found for the Pi-complexedArg43Gln E-PPase as well as for the mutant variantAsp117Glu of Y-PPase described previously.7

When P1 has left, two water molecules enter thispart of the active site, compensating for the loss ofthe initial ligands of the metal ions M3 and M4. Theside-chains of Arg43 and Tyr141 that directlyinteracted with P1 (Table 1) move substantiallyafter its departure (Figure 3(a)). The guanidinegroup of Arg43 preserves its orientation within theactive site cavity in all presently described struc-

Pase structures: (a) MnPPi–F–E-PPase; (b) MnPPi/Pi–F–E-

Figure 2 (legend on previous page)


tures. This finding suggests that the rotation of thearginine side-chain outside the active site that wasobserved earlier may not have been related to therelease of P1. The side-chain of Glu145 also shiftsfollowing the motion of Arg43, with which it formsan ion pair. This movement is transmitted along achain of H-bonded residues (not shown in thisFigure) to Gln133 of an adjacent trimer. Furtherprotein residues exhibiting alternative positions inthe Pi or PPi-containing complexes include Ser46,Lys148, and other residues that are supposed to playan important role in the interaction betweendifferent subunits of E-PPase.The residues Lys29, Glu31, and Asp42 do not have

double positions, but they occupy different locations(the atomic coordinates shift by more then four timesthe average coordinate error) depending on whetherPPi or Pi is bound in the active site (Figure 3(b)).Concerted rotations of the planes of the Glu31 andAsp42 carboxylate groups are observed that followthe motion of the M4 metal ion from the M4pp to theM4p position. The amino group of Lys29 and the

guanidine group of Arg43 are also involved in thismovement. All these residues belong to the sameβ-sheet, which is formed by two antiparallel strandsincluding residues 28–32 and 39–44. This structuralelement appears to move as a whole. The concertedconformational movements that are mediated byAsp42 most likely are required to properly positionLys29 and Arg43 for the release of the secondphosphate.8 Lys104 is another residue whose posi-tion differs in PPi and Pi-complexed structures(Figure 3(b)). An unusual feature, which was notobserved previously, is the involvement of theamino group of this lysine in Pi binding (Table 2).

Release of the P2 phosphate

Akey feature of the high-resolution structure of thephosphate complex of E-PPase is that only electro-philic phosphate P2 was found in the active site afterhydrolysis of PPi. It moves towards the entrance ofthe active site and occupies a similar location as thephosphate complex described earlier for theY-PPase4

Table 1. Distances from the metal ions and the oxygenatoms of PPi to ligands in the PPi-containing structures

Atom/metalion position Ligand

Distances (Å)

MgPPi–F–E-PPase

MnPPi–F–E-PPase

PPi-conformationof MnPPi/

Pi–F–E-PPase

M1 Asp65 Oδ1 1.9 2.19 2.20Asp70 Oδ2 2.15 2.15 2.15Asp102 Oδ1 2.06 2.18 2.12

F− 2.03 2.16 2.16Wat 322 2.25 2.18 2.21

M2 Asp70 Oδ1 2.19 2.18 2.13F− 1.93 2.15 2.28

Wat 315 2.06 2.07 2.18Wat 308 2.07 2.15 2.17Wat 353 2.10 2.28 2.30

M3 Asp97 Oδ2 1.98 2.14 2.08Asp102 Oδ2 2.13 2.10 2.12Wat (*Cl−)

3552.01 2.35* 2.50*

Wat 364 2.14 2.21 2.27M4pp Glu31 Oε2 2.09 2.28 2.30

Wat 480 2.08 2.30 2.27Wat 479 2.01 2.10 2.44Wat 378 2.01 2.30 2.29

PPi O1 M3 1.97 2.11 2.06Lys142 Nξ 2.81 2.93 3.11Wat 364 2.98 3.16 3.15Wat 361 3.05 3.08 2.94

O2 Tyr141 OH 2.74 2.68 2.62Arg43 NH2 2.85 2.86 3.00Wat 350 2.97 2.92 2.88

O3 M4 1.97 2.15 2.12Arg43 NH1 2.94 2.94 3.01Wat 480 3.21 3.16 3.22Wat 378 2.93 3.19 –

O Lys29 Nξ 2.99 3.06 3.01Wat 350 2.97 2.97 2.75

O4 M1 1.98 2.21 2.14M3 2.24 2.18 2.25

Wat 364 2.90 3.06 2.93O5 M2 2.06 2.17 2.06

Tyr55 OH 2.70 2.62 2.80Wat 353 2.80 2.80 2.99

O6 M4 2.02 2.12 2.12Asp67 Oδ2 2.99 2.99 2.68Wat 479 2.88 2.90 3.12Wat 378 2.88 3.18 2.99

Distances are shown in Å. All partners within 3.4 Å distances areindicated. Distances of M–O(PPi) bonds are included in the PPisection.

Table 2. Distances from the metal ions and the oxygenatoms of Pi to ligands in the Pi-containing structures

Atom/metalion position Ligand

Distances (Å)

Pi-conformationof MnPPi/

Pi–F–E-PPaseMnPi–E-PPase

MnPi–Arg43GlnE-PPase

M1 Asp65 Oδ1 2.20 2.23 2.17Asp70 Oδ2 2.15 2.17 2.11Asp102 Oδ1 2.12 2.17 2.09Wat 322 2.21 2.03 2.20

M2 Asp70 Oδ1 2.13 2.07 2.12Wat 308 2.18 2.21 2.18Wat 315 2.18 2.12 2.16Wat 353 2.16 2.27 2.14

M3 Asp97 Oδ2 2.08 2.14 2.14Asp102 Oδ2 2.12 2.14 2.12Cl− 355 2.50 2.34 2.55Wat 364 2.27 2.52 2.28Wat 416 2.26 2.21 2.21

M4p Glu31 Oε2 2.28 2.17 2.21Wat 377 2.21 2.07 2.20Wat 378 2.34 2.04 2.38Wat 356 1.92 2.34 2.19Wat 358 2.11 2.04 2.33

P2 O M4p 2.18 2.18 2.17Lys29 Nξ 3.16 3.14 2.98Wat 358 2.95 3.01 2.64Wat 416 2.62 2.81 2.72Wat 378 2.95 2.61 2.91

O4 M2 2.49 2.19 2.37Tyr55 OH 2.46 2.55 2.58

O5 M1 2.29 2.29 2.30M2 2.22 2.35 2.35M3 2.14 2.18 2.15

Lys104 Nξ 3.32 3.17 3.15Wat 364 3.15 3.26 3.13Wat 416 3.02 3.04 3.03

O6 M1 2.26 2.33 2.21M2 2.23 2.28 2.27

Wat 356 2.62 2.52 2.64Wat 315 3.44 3.15 3.34Wat 353 2.91 3.38 3.29

All partners within 3.4 Å distances are indicated. Distances of M–O(Pi) bonds are included in the Pi section.


where both Pi groupswere still bound to enzyme.Weobserved two different conformations of the P2phosphate, P2down and P2up, corresponding to thedifferent states of the active site. P2down relates to thestate immediately after hydrolysis, P2up to a subse-quent stage of catalysis. Double positionswere foundalso for the P1 phosphate, the M4 manganese ion,and some active site residues, but their unambiguousattribution to one or another state of the active sitewas rather difficult. The conformation of the P2phosphate in E-PPase described here is closelysimilar to P2down in Y-PPase, the orientation of theoxygen atoms and their coordination being the samein two enzymes (Figure 5). However, unlike Y-PPase,the X-ray structure trapped a stage of catalysis whenthe phosphate P1 had already been released from theactive site. A joint interpretation of the X-ray data

and the results of solution kinetic studies suggest thatP1 leaves first after the Mn-supported hydrolysis byE-PPase, while release of P2 is the rate-limiting stepas it was shown for the yeast enzyme.9 This structurediscloses a principally new aspect of the problem ofproduct release. Itwas proposed on a theoretical basethat the rotation of the P2 phosphate is necessary forits release so that the Onu oxygen atom would bedetached from the metal ions M1 and M2 and P2would be brought to the P2up position. Taking thefinding that P2 still adopts the P2down conformation inMnPi–E-PPase when P1 has already left into account,wemay suppose that this rotation of P2 is the slowestevent in the course of the hydrolytic reaction, and thatitmay be regarded as being responsible for the slowerspeed of hydrolysis when using Mn2+ as an activatoras compared to Mg2+.

Comparison of Mg2+ and Mn2+ as activators ofE-PPase

Hydrolytic activity of E-PPase can be supportedby either of these two cations. In both cases, the

Figure 3. The changes in the active site along the course of hydrolysis. (a) The active site of MnPPi/Pi–F–E-PPase.Shown are PPi conformation (red); Pi conformation (green); residues and ligands with similar positions in bothconformations (yellow). (b) Consecutive movement of the residues Lys29, Glu31, Asp42, Asp42, Lys104, Asp67, and theM4 metal ion. The structures shown are: MnPPi/Pi–F–E-PPase (blue), MnPi–EPPase (dark red), MnPPi–F–E-PPase(green).


activity profile versus cation concentration goesthrough a maximum revealing that there is aninhibitory binding site previously considered to beM4. The inhibition of E-PPase by an excess of themetal activator probably has a regulatory function invivo. Mn2+ shows a much higher affinity for theinhibitor site as compared to Mg2+; this finding wasproposed to account for the lower effective rates ofMn-supported hydrolysis, although this cationpotentially can be a stronger activator.10 As men-tioned above, the active sites of the structures ofMgPPi–F–E-PPase versus MnPPi–F–E-PPase werenearly identical (Figure 1), and the very substantialdifference in the activator efficiency of Mg2+ andMn2+ could not be explained on this basis. The twocations, however, behaved differently with respectto the state of E-PPase trapped in the X-ray studyafter PPi hydrolysis. Washing out fluoride from aMnPPi-F–E-PPase crystal made it possible to trapconsecutive intermediates, as described above,whereas the same procedure applied to a MgPPi–

F–E-PPase crystal only produced one intermediatestructure with a partly occupied pyrophosphate siteand the final structure of the holoform, i.e. E-PPasecomplexed with two metal ions (unpublished data).This finding may be explained under the assump-tion that M4p is the actual position of the inhibitorbinding site. The structural data obtained hereshowed that Mn2+ is capable of tight binding atposition M4p and thereby locking the P2 phosphatein the P2down state. Probably, high affinity for M4p isa key reason for the strong inhibition of E-PPase byan excess of Mn2+ but not by Mg2+. Under theconditions of our crystallization experiments, Mg2+

appears to be incapable of occupying the M4p

position, causing both phosphate groups to leavetoo fast for the intermediates to be trapped.

Possible role for Asp67/117 in PPase catalysis

The ternary enzyme–substrate–fluoride complexis rather weak in the case of E-PPase, and the active

Figure 4. Inhibition constant for fluoride, Ki, as afunction of pH for the Mn2+ ((1) left ordinate axis) or theMg2+-supported ((2), (3), right ordinate axis) hydrolysis ofPPi by the wild-type E-PPase ((1), (2) and Asp67Asnvariant (3)). Each point is an average of two independentexperiments. Standard deviations are indicated with errorbars. Inset: Dixon–Webb plot for the Mn2+-supportedhydrolysis. Continuous lines for the curve (1) and inset aredrawn corresponding to a single ionization model usingthe following parameters: pKa=7.7, Ki,max=7.3 mM. Thedotted line in the inset shows the theoretical titration curvewith a slope of 1.0. Continuous lines for the curves (2) and(3) are non-linear regression fits to the built-in Hill equa-tion (SigmaPlot). Midpoint pH values are pH 9.1(±0.2) andpH 9.3(±0.1), respectively.


enzyme can easily be recovered from it by dilution orby washing out F−. The only mutant variant thatshowed significant stabilization of this complex wasAsp67Asn.6 This may signify that the carboxylicgroup of Asp67 participates in the release of F− fromthe active site when the inhibitor is washed out. Inthe structures of PPi-complexes of PPases, F−

occupies the location of the attackingwater moleculeWa that after hydrolysis converts into the oxygenatom Onu of the P2 phosphate. Therefore, thestabilization of its binding by a mutation of Asp67suggests that the carboxylic group of Asp67 permitsthe detachment of Onu from the coordination spheresof the metal ions M1 and M2 and thus facilitates therotation of P2 into the P2up position. The series ofstructures described here shows that the side chain ofAsp67 changes its orientation depending onwhetherPPi or Pi is bound to the active site (Figure 3(b)).Taken together, these results enable us to propose

a role for Asp67 in the course of catalysis, asillustrated by the scheme shown in Figure 6. Of thesix theoretically expected intermediates shown inFigure 6, four (denoted byA, B,D, and E) have beenconfirmed by X-ray data for either E or Y-PPase. Inthe active site of the EPPase complexed with Mg2+

the carboxylate of Asp67 is involved in the secondcoordination sphere of M2 (A). After binding thesubstrate, the metal ions M1 and M2 are broughtcloser to each other, and Asp67 is liganded with thebridging water molecule Wa (MgPPi–F–E-Ppase; B).As the system relaxes further, a proton has to beejected from Wa, resulting in the formation of theattacking nucleophile OH− that repulses the carbox-ylate of Asp67 (C). Subsequently, hydrolysis of PPioccurs including the attack of OH− on the electro-

philic phosphorus atom, breakdown of the P–Obond, reversal of the P2 configuration, and conver-sion of OH− into Onu. After departure of P1, Mn2+

occupies the M4p binding site, so that one of itswater ligands interacts with Asp67p and simulta-neously with Onu (D; down conformation of P2,MnPi–E-PPase). When the side-chain of Asp67relaxes to the position Asp67pp (E), it pushes thiswater molecule so that it displaces Onu of thephosphate P2. As a result, P2 is brought into theup position. After P2 is released (F), the active siterelaxes to the initial state A.Thus, in terms of the role of Asp67, this scheme

assumes that Asp67 polarizes the O–H bond in Waand thereby facilitates the release of H+ (step 2). Therotation of the carboxylate of Asp67 into the positionAsp67p and back permits a new water molecule tosubstitute for Wa and to assume a proper positionfor the next catalytic cycle (step 4). This motion alsocauses P2 to rotate from the down into the upposition, which is more favourable for leaving. Asmentioned above, this rotation may be regarded as akey event in the release of the P2 phosphate afterMn-supported hydrolysis.The general scheme proposed here appears to be

valid also for Y-PPase. The intermediate B can beobserved in the structure of PPi-complexed Y-PPase(1E6A4). Two positions of Asp117 (corresponding toAsp67 in E-PPase) were also observed in Y-PPasecomplexedwith two Pi groups (1E9G

4). In contrast toE-PPase, however, the carboxylate of Asp117 in thiscase shifted only slightly, remaining in H-bond inter-action with F−/Onu both in the up and down con-formations (D and E in Figure 6). Such a difference inthe conformational changes experienced by theAsp67/117 side-chain might be partly responsiblefor the different behaviour of the two enzymes withrespect to fluoride inhibition. Within this scheme, F−

can replace the water molecule bridging M1 and M2in the intermediatesB or E. In the case of E-PPase, thebinding of anions to either of the metal ions mustprovoke an electrostatic repulsion of Asp67 and itsrotation into the position Asp67p. If the fluoride con-centration in themediumdecreases, F−may easily bereplaced with a water molecule by the same mecha-nism as proposed for Onu. In the mutant variantAsp67Asn, the side-chain is uncharged, and there isno repulsive interaction of Asn67 and F−. Hence, thefluoride ion remains H-bonded to the asparagine,and the entire enzyme—substrate–inhibitor complexis more stable than in case of the wild-type E-PPase.In the case of Y-PPase, the carboxylate of Asp117cannot move away from F− due to steric constraints;4

as a result, it remains protonated in order to avoid anenergy penalty for holding together two negativeparticles, and the enzyme—substrate–fluoride com-plex again is very stable.

pH-effects of fluoride inhibition and ionisation ofAsp67

Since the likely role of Asp117 is to accept protonsfromWa to form the attacking nucleophile OH−,4 two

Figure 5. Superposition of the active sites of YPPase (red/black) and E-PPase (yellow/gray) complexed with MnPPi(a) or MnPi (b).


possible protomers are shown in Figure 6 for theintermediate B. This assumption is supported by thefact that all X-ray structures of PPi or Pi-complexed Y-PPase show the carboxylate of Asp117 liganded toWa/Onu. In the case of E-PPase, however, the mutantvariant Asp67Asn retains measurable hydrolyticactivity despite the loss of presumable acceptorproperties.11 In addition, the structure of PPi-com-plexed E-PPase described here reveals that no proteinacceptor is needed for the deprotonation of Wa. Thewater molecule Wa is located within the sphere ofinfluence of a chain of H-bonded water molecules.Thus, a proton liberated from a weakened H-O bond

in Wa may be transported to the protein surface,where it will be taken up by buffer anions (Figure 7).The proton transport may proceed according to theGrotthuss relay mechanism12 via chemical exchangeof hydrogen nuclei along the chain of water mole-cules. The first water molecule in this chain is equi-distant from Wa and the Oδ2 atom of Asp67. Hence,the translocation of H+ from Wa does not necessarilyimply its transfer to the carboxylate of Asp67.All water molecules that form the chain are

precisely positioned through tight H-bond contactswith protein groups. Since all protein residues thatinteract with this water chain (Figure 7) are in-

Figure 6. Participation of Asp67 in the catalytic cycle of EPPase. The arrangement of Asp67 and the related active siteresidues is shown schematically. Metal ions are indicated by grey circles. The intermediates A, B,D and E are based on X-ray data; intermediates C and F are proposed on a theoretical basis. A, Holo-E-PPase (1obw). B, After substrate is bound.Asp 67 in the Asp67pp position (MgPPi–F–E-PPase). Two possible protomeric forms for the pair Wa-Asp67 are shown. C,Theoretically predicted repulsion of Asp67 after the proton is emitted from Wa. Two alternative conformations of Asp67can be actually seen in MnPPi/Pi–F–E-PPase. D, After P1 is released. P2 in the down conformation, Asp 67 in the Asp67p

position (MnPi–E-PPase). E, Concerted rotation of Asp67 back into the Asp67pp position and P2 into the “up”conformation (1e9g10). F, Holo-E-PPase after P2 is released.


variant in the sequences of the family I PPases,except for Gly66, one may propose that this protontransport system may be common to theseenzymes. Indeed, some equivalent water moleculesthat interact with corresponding residues are alsopresent in the PPi-complexed Y-PPase (1E6A);however, some of these water molecules are morethan 4.5 Å apart and do not form a continuouschain. We found another chain instead, startingfrom the Oδ2 atom of Asp117 and reaching thesurface near Lys238. It appears, however, that thischain may not play the same role in protontranslocation in Y-PPase, since the first watermolecule is H-bound to Asp117 but not to Wa.Thus, ionization of Wa in this case may require thatthe liberated proton is taken up by the carboxylateof Asp117. Such a difference in the protontranslocation mechanism may account for part of

the difference in the properties of the two enzymes.Based on this comparison one may suggest that thewater chain observed in the bacterial enzyme lostits importance as the proton transport pathway,due to evolutionary changes in the surface of thePPase globule.The strong pH-dependence of Ki for fluoride

inhibition of Mn-supported hydrolytic activity ofE-PPase corresponded to a classical single-ionizinggroup model. We had expected that an analysis ofthis pH-profile would enable us to estimate the pKavalue of the carboxylic group of Asp67 as the onlytitratable protein ligand of Wa (another groupinteracting with fluoride/Wa is Asp65, which iscoordinated to the metal ion M1 and thereforethought to be ionized in the tested pH range). Ourstudy, however, showed that mutations of the metalliganding groups including Asp67 did not affect the

Figure 7. H-bonded water molecules form a chain capable of transferring H+ from Wa to the protein surface. TheFigure shows the metal ions M1 and M2, the bridging water molecule Wa, and the active site residues that form H-bondsto water molecules in the chain. All distances between water oxygen atoms in the chain are within 2.9 Å. The chain ispresent both in the MgPPi–F–E-PPase and MnPPi–Fm–E- Continuous PPase structures.


pH transition of Ki. This fact together with the lowslopes of the double-logarithmic plots for Mg-supported hydrolysis may indicate the complexcharacter of the apparent inhibition constant. Alter-natively, pH may affect fluoride binding by chang-ing the metal content of the enzyme or the amount ofenzyme–substrate complex. This supposition, how-ever, seems unlikely since neither the metal bindingaffinities nor Km change much over the pH range pH8–10 where the largest difference in Ki was observedfor the Mg–enzyme complex.One of the possible factors affecting the observed

pH-dependence might be a possible ionization ofWa. Assuming that the apparent Ki value may bedefined not only by the affinity of the enzyme for F−,but also by the affinity for Wa/OH−, one mayspeculate that it is easier for fluoride to displaceuncharged water molecules weakly attached to the

Table 3. Data collection and refinement statistics

MnPPi–F–E-PPaseMnPPi/

Pi–F–E-PPase

Unit cell (Å) a=b=109.52c=75.59

a=b=109.37c=75.08

Unique reflections(Total observation)

46,439 (442,448) 56,152 (528,300)

Resolution (Å) 20.0–1.30 (1.32-1.30)a 20.0–1.20 (1.22-1.2Completeness (%) 99.2 (100.0) 99.9 (100.0)

<I/σ(I)> 19.0 (2.0) 16.1 (2.54)Rmerge(%) 4.9 (53.8) 4.7 (41.4)R/Rfree (%) 14.8/17.3 12.8/15.2No. of non-hydrogenprotein atoms

1380 1380

No. of water molecules 253 283Average B-factor (Å2) 30.7 21.3r.m.s.d. bond length (Å) 0.018 0.018r.m.s.d. bond angle (Å) 0.042 0.039Ramachandran plotResidues in most

favourable region/additional allowedregion (%)

92.7/7.3 92.7/7.3

Esd of atomic coordinates 0.047 0.043a Values in parentheses are for the highest-resolution shell.

metal ions M1 and M2 than to displace the chargedOH−. Under this assumption, the ionization of Wamay strongly depend on the nature of the metalactivator, possibly explaining the fact that themidpoint for the pH transition of Ki is significantlylower for Mn2+ than for Mg2+. This might accountfor the higher potential efficiency of Mn2+ asactivator of the hydrolysis as compared to Mg2+.

Materials and Methods

Crystallization

Recombinant E. coli PPase was isolated and purified asdescribed.13 Mutant variant Arg43Gln was obtained withthe procedure developed for the other mutant variants ofE-PPase.7 Protein crystals were grown by the hanging

MnPi–E-PPase MgPPi–F–E-PPaseMnPi– Arg43Gln

E-PPase

a=b=110.66c=73.97

a=b=109.36c=75.16

a=b=110.98c=73.05

22,184 (220,141) 47,389 (625,534) 94,372 (1,591,040)

0) 20–1.65 (1.68–1.65) 20–1.22 (1.23–1.22) 20–1.05(1.07–1.05)99.9 (99.7) 99.0 (99.2) 97.4 (96.1)16.7 (2.5) 15.9 (3.68) 17.2 (2.64)5.0 (46.0) 4.7 (33.8) 5.1 (20.6)16.5/20.9 14.8/17.3 12.5/15.5

1380 1380 1380

144 187 29936.4 36.1 18.80.017 0.017 0.0180.044 0.041 0.038

94.0/6.0 94.0/6.0 92.1/7.9

0.077 0.048 0.034


drop technique in 0.2 M sodium acetate buffer (pH 5.5),using 1.5 M–1.7 M NaCl as precipitant.14 The proteinconcentration in the drops was 7–8 mg/ml.Crystals of the PPase complexed with MgPPi and F−

were obtained by co-crystallization of the enzymewith 70–80 mM MgCl2, 30–45 mM KH2PO4, and 3 mM NaF. Thecrystals were soaked with solutions containing step-wiseincreased gradient concentrations of MgCl2 and KH2PO4up to their final values of 120mMand 60mM, respectively.For the crystals of PPase complexed with MnPPi and F−,

cofactors were added during co-crystallization including14 mM MnCl2, 7 mM KH2PO4, and 5 mM NaF. The finalfull-occupancy complex was obtained by soaking with7 mM MnCl2, 14 mM KH2PO4, and 10 mM NaF. TheArg43Gln mutant variant complexed with Pi was obtainedunder the same conditions.

Data collection and structure refinement

The X-ray diffraction data were collected on the beam-line BW6 (DESY, Hamburg, Germany) using synchrotronradiation at a wavelength of 0.906 Å. A MAR-CCD wasused as a detector. The crystals were cooled to 100 K usingglycerol as cryoprotector.5 After collection of an initialdiffraction data set from a crystal of the full-occupancycomplex of E-PPase with MnPPi and F−, the crystal wassoaked in a cryosolution without F−.5 Subsequently, thecrystal was flash-cooled again, and a new data set wascollected. This cyclic procedure permitted us to collect fulldata sets corresponding to several different structuresfrom one and the same crystal. The optimum choice of thenumber of cycles and of the time spent for each cycle wasestablished experimentally. Six minutes were sufficient tohydrolyze 50% of PPi in the PPase, while completehydrolysis took about 15 min. The overall data collectiontime and the resolution limit were optimized on the basisof preliminary data using the BEST program.15

The PPase crystals belonged to space group R32 andcontained one monomer in the asymmetric unit. Thediffraction data were processed and scaled using theDENZO and SCALPACK program packages.16

Refinement was performed with the program SHELX.17

The structure of CaPPi-EPPase was used as an initialmodel. PROCHECK18 showed good geometries withoutoutliers for all refined structural models. The data col-lection and refinement statistics are summarized in Table 3.

Chemicals

The chemicals used in the study included in particularATP-sulfurilase, luciferase, adenosine phosphosulphate,luciferin, dithiothreitole, HClO4, H3PO4, Tris, Mes (Fluka,Switzerland), Hepes and Capso (ICN, USA). Otherchemicals were purchased in high-purity grade fromFluka, Serva, Merck or Pharmacia Fine Chemicals. Allstock solutions were freshly prepared with high qualitywater purified in a MilliQ column.

Kinetic measurements

The hydrolytic activity of PPase was determined by therate of Pi release from MgPPi or MnPPi. A semi-automaticphosphate analyser was used for quantitative Pidetermination.19 Hydrolysis was carried out at 25 °Cand at the device sensitivity of 10 μM Pi for the full-scale.Inhibition of hydrolysis by fluoride was studied with

50 μM substrate in 0.05 M buffer of corresponding pH

containing 5 mM free Mg2+ or 50 μM free Mn2+. Theconcentration of NaF was varied between 0–0.5 mM forMg2+-supported hydrolysis and 0–20 mM for Mn2+-supported hydrolysis, respectively. The inhibition constantKi for fluoride was determined by fitting the experimentaldependence of activity to fluoride concentration tohyperbola using non-linear regression (SigmaPlot). Thebuffers used were Mes–NaOH, Mops–NaOH, Hepes–NaOH, Tris–HCl, and Capso–HCl, in the pH ranges 5.8–6.7, 6.5–7.5, 7.0–7.5, 7.2–9.0, and 9.0–10.0, respectively.

Solution study of synthesis of PPi in the active site

Reaction mixtures of total volume 50 μl containing2–6 mg/ml enzyme in 0.2 M Tris–HCl (pH 8.0), 50 μMEGTA, 10mM freeMg2+, and 0.5 mMNaF, were incubatedfor 30 min at 25 °C in the presence of 0–20 mMmagnesiumphosphate. A second set of mixtures contained all thesame components excluding NaF. The reaction wasinitiated by addition of enzyme and stopped by additionof 10 μl of 5 M triflouroacetic acid. After 10 min, theprecipitate was separated by centrifugation for 10 min at10,000 rpm. The amount of pyrophosphate in the 5 mlportion of supernatant was measured.20 The experimentwas performed three times at each concentration of MgPi.All chemicals were first checked for the absence of PPi.

Protein Data Bank accession numbers

Coordinates of MgPPiF-EPPase, MnPPiF-EPPase,MnPPi/Pi-F-EPPase, MnPi-EPPase and Arg43Gln-EPPasewere deposited in the Protein Data Bank, RCSB, withaccession codes 2AUU, 2AU9, 2AU6, 2AU7 and 2AU8.

Acknowledgements

This work was supported by the Russian Foun-dation for Basic Research (grants 06-04-49127 and1706-2003-4), and by INTAS (grant YSF-2002-0374).

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Edited by R. Huber

(Received 31 July 2006; received in revised form 23 November 2006; accepted 29 November 2006)
Available online 2 December 2006

reversible inhibition of escherichia coli inorganic pyrophosphatase by fluoride: trapped catalytic...

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