Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase

Eur. J. Biochem. 267, 7065±7074 (2000) q FEBS 2000

Characterization of active-site mutants of Schizosaccharomyces pombephosphoglycerate mutaseElucidation of the roles of amino acids involved in substrate binding and catalysis

Jacqueline Nairn1, Doris Duncan1, Naomi E. Price1, Sharon M. Kelly1, Linda A. Fothergill-Gilmore2,Stanislava Uhrinova3, Paul N. Barlow3, Daniel J. Rigden4 and Nicholas C. Price1

1Department of Biological Sciences, University of Stirling, Scotland, UK; 2Department of Biomedical Sciences, University of Edinburgh,

Scotland, UK; 3Edinburgh Centre for Protein Technology, University of Edinburgh, Scotland, UK; 4Bioinformatics Laboratory,

Universidade CatoÂlica de BrasõÂlia, Brazil

The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from

the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids

examined could be divided broadly into those presumed from previous related structural studies to be important

in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120

and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants

except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm::HIS) in good

yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A

mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and

characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by

guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate

(3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration

calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic

cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the

enzyme using multidimensional NMR techniques.

Keywords: catalytic mechanism; phosphoglycerate mutase; protein stability; site-directed mutagenesis; substrate

binding.

The enzyme phosphoglycerate mutase (GriP mutase; EC 5.4.2.1)catalyses the interconversion of 2-phosphoglycerate and 3-phos-phoglycerate. There are two structurally and kinetically distinctclasses of GriP mutase: those that require the cofactor 2,3-bisphosphoglycerate (Gri2,3P2) for activity and those that donot. These are termed dependent GriP mutase (dGriP mutase)and independent GriP mutase (iGriP mutase), respectively [1].dGriP mutases occur in vertebrates, yeast and some eubacteria,whereas iGriP mutases are found in higher plants, nematodes,archea and other eubacteria. The information available on iGriPmutases suggests that this enzyme is a member of the alkalinephosphatase superfamily, may proceed via an intramoleculartransfer of the phospho group, and is metal ion dependent[2±6].

By contrast, dGriP mutase is part of the acid phosphatasesuperfamily which includes fructose-2,6-bisphosphatase(Fru2,6P2ase), pyrophosphatase and acid phosphatase [1]. Of

the dGriP mutases, the enzyme from Saccharomyces cerevisiaehas been the most extensively characterized [7±11]. In thecatalytic cycle, dGriP mutase catalyses the intermoleculartransfer of phospho groups, and Gri2,3P2 is required to donatea phospho group to form a covalent phosphohistidine inter-mediate in the active site, thereby `priming' the enzyme. Asimilar catalytic mechanism also occurs in the structurallyrelated enzyme rat liver Fru2,6P2ase [12].

The crystal structure of S. cerevisiae dGriP mutase refined to0.212 nm, together with the modelling of Gri2,3P2 bound todGriP mutase suggested the mode of binding of the cofactor/reaction intermediate and a possible mechanism of enzymephosphorylation [8]. The basis for the positioning of Gri2,3P2

in the modelled complex was the presence of two sulfatemolecules in the active-site cleft. Because the spacing of thesesulfates (0.73 nm) matched reasonably well the spacing of thetwo phospho groups in a relaxed conformation of Gri2,3P2

(0.61 nm), the Gri2,3P2 molecule was positioned with itsphospho groups overlaid upon the positions of the crystallo-graphically observed sulfates. With the 2-phospho groupoverlaid on the sulfate located closer to H8, the carboxylategroup of Gri2,3P2 was most favourably positioned to interactwith N14, T20 and K97. An equally attractive binding modecould be obtained with the 3-phospho group closer to H8 [8].The mechanism of dGriP mutase was thereby proposed toinvolve attack by the catalytically important H8 on the phosphogroup of Gri2,3P2, assisted by proton abstraction from the sidechain of E86 via an intervening water molecule [8]. Likewise,

Correspondence to N. C. Price, IBLS Division of Biochemistry and

Molecular Biology, Joseph Black Building, University of Glasgow,

Glasgow G12 8QQ, Scotland, UK. Fax: 1 44 141 330 6447,

Tel.: 1 44 141 330 2889, E-mail: [email protected]

Abbreviations: GriP, 3-phosphoglycerate; Gri2,3P2,

2,3-bisphosphoglycerate; Fru2,6P2ase, fructose-2,6-bisphosphatase;

GdmCl, guanidinium chloride; ITC, isothermal titration calorimetry;

HSQC, heteronuclear single quantum coherence.

Enzyme: phosphoglycerate mutase (GriP mutase; EC 5.4.2.1).

(Received 16 August 2000, accepted 4 October 2000)

X-ray crystallography, kinetic studies, site-directed muta-genesis and NMR work on the homologous enzyme Fru2,6-P2ase [12±14] have given a detailed description of thebisphosphatase active site and have led to a proposedmechanism for the Fru2,6P2ase reaction.

In the light of these recent mechanistic proposals for bothS. cerevisiae dGriP mutase [10] and rat testis Fru2,6P2ase [12],the roles of a number of dGriP mutase active-site residues needto be clarified. The 0.212-nm structure of S. cerevisiae GriPmutase suggests that R113 and R114 interact electrostaticallywith the 3-phospho group of the substrate. Likewise, thisstructure implicates R87 as interacting with substrate phosphogroups during catalysis. R7 and S55 also have putative roles

in catalysis, with R7 possibly binding to the carboxylate groupof Gri2,3P2 as it is transferred to H8, and with S55 contributingto the orientation of another active-site histidine (H181). Site-directed mutagenesis together with structural studies on bothS. cerevisiae GriP mutase [8] and rat liver Fru2,6P2ase [12,14]support the role of E86 as the proton donor/acceptor duringcatalysis.

Figure 1 shows the arrangement of these residues at theactive site of S. cerevisiae dGriP mutase. The structuralconsequences of phosphorylation of dGriP mutase remainunclear because it has so far proved impossible to crystallise thephosphorylated enzyme. It seems likely that phosphorylationis accompanied by structural changes, as exposure of crystals

Fig. 1. Active-site cleft of the dGriP mutase

from S. cerevisiae [8] showing a model of

bound Gri2,3P2. The model was constructed as

described in the text. The amino-acid bonds of

the protein are shaded to aid their differentiation

from the Gri2,3P2 molecule. Interactions of S55

with H181 and of Gri2,3P2 carboxylate with R7

are indicated by dotted lines. The figure was

constructed using molscript [39].

Fig. 2. Alignment of S. pombe and

S. cerevisiae dGriP mutase sequences showing

the active-site residues mutated in S. pombe

dGriP mutase. S. pombe and S. cerevisiae dGriP

mutase sequences [17] were aligned using

clustal w [40] with subsequent modifications of

the N-terminal and C-terminal regions to allow

direct comparison of these regions. The

active-site residues mutated in S. pombe dGriP

mutase are shown as underlined (R14, S62, E93,

R94, R120 and R121). Asterisks indicate residues

that are conserved in both sequences, colons (:)

indicate positions of strong similarity, and dots (.)

indicate positions of weak similarity. Both

S. pombe and S. cerevisiae numbering are given.

7066 J. Nairn et al. (Eur. J. Biochem. 267) q FEBS 2000

of S. cerevisiae dGriP mutase to Gri2,3P2 results in theircracking [8]. Nevertheless, the structural changes are likely tobe relatively small because (a) phosphorylation of S. cerevisiaedGriP mutase does not lead to any changes in the far-UV ornear-UV CD spectra [10] and (b) comparison of the crystalstructures of unphosphorylated [15] and phosphorylated [16]Fru2,6P2ase reveals only minor conformational differences.

Site-directed mutagenesis of Schizosaccharomyces pombedGriP mutase was used to study the roles of a number ofdGriP mutase active-site residues. The following residues weremutated: R94, R120, R121 (substrate binding), E93 (protondonor/acceptor) and R14 and S62 (phosphotransfer). Thecorrespondence between the numbering of these residues andthose of S. cerevisiae dGriP mutase is shown in the sequencealignment (Fig. 2). The particular interest in S. pombe dGriPmutase is that it is a monomer with a molecular mass of 23 kDa[17], which is within the size range for high-resolution NMRspectroscopy [18,19]. By use of multidimensional NMRtechniques applied to isotopically labelled enzyme, a backbonestructure (rms 0.10 nm) of the enzyme has been determined[19]. By contrast, S. cerevisiae dGriP mutase is a tetramer witha molecular mass of 110 kDa.

Here we describe the synthesis and characterization of anumber of active-site mutants (R14Q, S62A, E93D, R94Q,R120Q, R121Q) and the double mutant (R120,121Q) ofS. pombe dGriP mutase designed to probe the mechanism ofthis enzyme; this work complements and extends our earlierinvestigations on the histidine side chains of the enzyme [20].The mutants have been characterized in detail with respect tooverall structural properties (by CD), kinetic properties,stability of the phosphoenzyme, stability of the folded state,and ligand binding. In addition, the solution structure of theE93D mutant has been examined by multidimensional hetero-nuclear NMR and shown to be very similar to that of the wild-type enzyme [19].

Characterization of these mutants has given some insightinto the role of active-site residues in the mechanism ofS. pombe GriP mutase. The results not only corroborate therecent mechanism proposals for both S. cerevisiae GriP mutaseand rat testis Fru2,6P2ase, but should also provide a foundationfor the interpretation of ongoing NMR experiments intended toexplore the dynamics and solution structure of the enzymeduring its catalytic cycle.

M A T E R I A L S A N D M E T H O D S

Site-directed mutagenesis and overexpression of S. pombedGriP mutase was performed using the PCR method with thePGK-based vector pMA91 as detailed in Nairn et al. [20].

The mutagenic primers (altered sequences underlined) wereas follows:

R14Q primer: 5 0-GTCCTTACCCAACACGGTGAS62A primer: 5 0-GCCTTCACCGCTGCTCTTCAE93D primer: 5 0-CGAGAAGCTCAACGACCGTTACTACGR94Q primer: 5 0-CTCAACGAGCAATACTACGGTGR120Q primer: 5 0-GTCCAAATCTGGCAACGTTCTTATGR121Q primer: 5 0-TCTGGCGCCAATCTTATGACR120,121Q primer: 5 0-GTCCAAATCTGGCAACAATCTT-

ATGACATTGCThe replacement amino acids were chosen to attempt

to minimize the impact on the tertiary structure of the protein.After site-directed mutagenesis, both strands of the mutantgenes were sequenced. In addition, MS analysis of the mutantenzymes confirmed that the desired mass change had resulted.

Wild-type and mutant constructs (except for the R14Qconstruct) were transformed into the strain of S. cerevisiae(S150-gpm::HIS3) from which the chromosomal copy of thegene encoding S. cerevisiae GriP mutase has been deleted [9].The R14Q construct was transformed into the S. cerevisiaestrain AH22 (leu2-3 2-12 his4-519can1).

Overexpressed active-site mutants of S. pombe dGriP mutasewere purified by ion-exchange chromatography on DEAE-cellulose DE52 in an analogous fashion to S. cerevisiae dGriPmutase [9] followed by gel filtration on Sephacryl S200. TheSephacryl S200 column was eluted with 50 mm Tris/HCl,pH 8.0. Concentrations of GriP mutase were determinedspectrophotometrically using the value of 1.40 for the A280

of a 1-mg´mL21 solution in a cell of pathlength 1 cm. Thisvalue was calculated from the amino-acid composition of theenzyme [17,21]; concentrations determined in this mannerwere found to agree to within 5% with those determined bythe Coomassie blue-binding method of Sedmak and Grossberg[22].

The assays of mutase and phosphatase activities wereperformed as described by White and Fothergill-Gilmore [9].The mutase assay components included: 30 mm Tris/HCl(pH 7.0), 20 mm KCl, 5 mm MgSO4, 0.2 mm ADP, 0.15 mmNADH, 10 mm 3-phosphoglycerate (GriP), 0.2 mm Gri2,3P2,0.08 U´mL21 enolase and 0.5 U´mL21 each of pyruvate kinaseand lactate dehydrogenase. For the determination of the Km forGriP, the Gri2,3P2 concentration was maintained at 0.2 mm;for the determination of the Km for Gri2,3P2, the GriPconcentration was maintained at 10 mm. Kinetic parameterswere obtained by nonlinear regression fitting to the Michaelis±Menten expression using the program leonora [23]. Phos-phatase activity was measured using similar conditions with theexception that 30 mm Tris/HCl was at pH 8.0 and GriP wasomitted.

The CD spectra of wild-type and mutant GriP mutaseswere recorded in a Jasco J-600 spectropolarimeter at 20 8C,calibrated with reference to 1S-(1)-10-camphorsulfonic acid.Spectra in the far-UV region (260±195 nm) were recordedin cylindrical cells of path length 0.02 cm, using proteinconcentrations in the range 0.3±0.5 mg´mL21. Spectra inthe near-UV (320±260 nm) were recorded in a cell ofpathlength 0.5 cm, using protein concentrations in the range1.1±1.3 mg´mL21. In each case, four scans (recorded at a scanrate of 10 nm´min21 with a time constant of 2 s) were averagedand corrected by subtraction of a spectrum of buffer alone.Mean residue ellipticities were calculated using a value of112.4 for the mean residue weight of the enzyme [17].

Fluorescence spectra were recorded on a Perkin±Elmer LS50spectrofluorimeter at 20 8C, using cuvettes of 1 mL capacity,at a protein concentration of 0.1 mg´mL21. In each case,four scans (recorded at a scan rate of 50 nm´min21) wereaveraged, and corrected by subtraction of a spectrum ofbuffer alone.

The stability of the folded state of each protein towardsGdmCl denaturation was determined as outlined by Pace [24].Concentrations of solutions of GdmCl (Ultrapure) werechecked by refractive index measurements [24]. After incu-bation in the presence of a given concentration of GdmCl in50 mm sodium phosphate buffer, pH 8.0, for 1 h at 20 8C, theextent of unfolding of each protein (0.1 mg´mL21) wasassessed by changes in fluorescence intensity at 350 nm, withexcitation at 290 nm, and by changes in ellipticity at 225 nm.Unfolding curves were analysed according to the two-statemodel [24], using linear extrapolation to the ordinate to obtain

q FEBS 2000 Active-site mutants of phosphoglycerate mutase (Eur. J. Biochem. 267) 7067

the free energy stabilization of the folded state in the absence ofGdmCl.

Positive ion electrospray mass spectrometry was performedon a Platform II mass spectrometer (Micromass UK Ltd,Manchester, UK). The samples were infused at a rate of8 mL´min21, and the ions produced in an atmospheric pressureionization/electrospray ion source. The source temperature was65 8C, and the drying and nebulizing gas flow rates were 300and 20 L´h21, respectively. A potential of 3.5 kV was appliedto the probe tip, and a cone voltage gradient optimized for thesamples was used. The quadrupole mass analyser was scannedat 100 atomic mass units´s21 after appropriate externalcalibration. The acquisition and deconvolution of data wereperformed using a MaxLynx (Version 2.3 Build 005) WindowsNT PC data system, utilizing the Maximum Entropy Electro-spray software algorithm. The mass accuracy of all measure-ments was within 0.1±0.5 on the m/z scale.

Isothermal titration calorimetry (ITC) experiments tomeasure the binding of GriP to S. pombe GriP mutase (wild-type, E93D and R120,121Q) were performed at 25 8C using aMicrocal VP-ITC titration microcalorimeter following standardinstrumental procedures [25,26] with a 250-mL injectionsyringe and 320 r.p.m. stirring. S. pombe GriP mutase wasdialysed extensively against buffer (30 mm Tris/HCl, pH 8.0)and degassed gently immediately before use. GriP wasdissolved in the same dialysis buffer, and the pH adjustedwhere necessary. Protein concentrations in the ITC cell weredetermined from UV absorbance measurements at 280 nmusing the value of 1.40 for a 1-mg´mL21 solution in a cell ofpathlength 1 cm. A typical binding experiment involved aninitial 1-mL injection, followed by 25 � 10 mL injections of1 mm GriP into the ITC cell (< 1.4 mL active volume)containing 40±80 mm (0.95±1.9 mg´mL21) S. pombe GriPmutase. Control experiments were performed under identicalconditions by injection of ligand into buffer alone (to correctfor heats of ligand dilution) and injection of buffer into theprotein solution (to correct for heats of dilution of the protein).Integrated heat effects, after correction for heats of dilutionwhere necessary, were analysed by nonlinear regression interms of a simple single-site binding model using the standardMicrocal origin software package. For each thermal titrationcurve, this yields estimates of the apparent number of bindingsites (N) on the protein, the association constant andthe enthalpy of binding. In cases of weak ligand bindingthe titration curve is too gradual to allow unambiguousestimation of N, and in such cases the stoichiometry may befixed at N � 1 for regression fits. Other thermodynamicquantities were calculated using standard expressions, i.e.DG8 � ±RTlnK � DH8 2 TDS8.

The 15N-labelled S. pombe GriP mutase sample [19] forthe 2D heteronuclear single quantum coherence (HSQC)experiment was prepared as a 0.6-mm solution in 0.6 mLH2O/D2O (90 : 10), containing 0.2 m sodium acetate and 0.4 m(NH4)2SO4 at pH 6.4. The 2D 1H-15N HSQC NMR spectrum[27] was recorded at 37 8C on a Varian Inova-600 NMRspectrometer equipped with a 5-mm triple resonance probe.The spectral width was 10 500 Hz in the acquisition dimension(1H) and 2225 Hz in the indirect (15N) dimension. The size ofthe acquired matrix was 2048 � 128 complex data points. ALorentzian-to-Gaussian window function was used for reso-lution enhancement in the acquired dimension and the sine-bellsquare function in the indirect direction, followed by zerofilling.

Examination of crystal structures and modelling of possibleGri2,3P2-binding modes were carried out using O [28].

R E S U LT S A N D D I S C U S S I O N

Expression and purification of S. pombe GriP mutasewild-type and active-site mutants

All mutant and wild-type GriP mutases were expressed inS. cerevisiae (S150-gpm::HIS) as described in Nairn et al. [20]with the exception of the R14Q mutant. The pMA91.R14Qconstruct was unable to complement this GriP mutase-deficient

Fig. 3. Spectroscopic properties of wild-type S. pombe GriP mutase and

mutant GriP mutases. Spectra for the wild-type, R14Q, E93D and R120Q

mutants are shown as solid lines, dashed lines, dotted lines and dotted/

dashed lines, respectively. Panels (a) and (b) show far-UV CD spectra and

near-UV CD spectra, respectively. Panel (c) shows the fluorescence

emission spectra with excitation at 290 nm.


S. cerevisiae strain to permit selection on glucose. The R14Qmutant was obtained by transforming S. cerevisiae strain AH22with the pMA91.R14Q construct. High levels of expression ofS. pombe wild-type and mutant GriP mutases were achieved,in particular for some of the less active mutants which wereoverexpressed in larger amounts to complement the GPM-deficient S. cerevisiae strain and permit growth on glucose. Allmutants except S62A were readily purified to homogeneity byion-exchange chromatography followed by gel filtration aspreviously described in Nairn et al. [20]. The gel-filtrationstep also allowed the R14Q mutant expressed in strainAH22 to be separated from the much larger S. cerevisiaedGriP mutase. The yield of purified protein ranged from< 20 mg´L21 of culture (for wild-type, R120Q, R121Q andR120,121Q) to 60 mg´L21 for some of the less active mutants(E93D, R94Q).

S62A was successfully overexpressed; however, on purifi-cation, the activity of this mutant was rapidly lost, implying thatthe substitution of S62 with an alanine residue rendered theprotein unstable. It is possible that the substitution leads tothe formation of a cavity in the structure, thus generating asource of instability. A second possibility is that S62 isimportant in maintaining the orientation of H163 of the activesite. The side chains of the equivalent S. cerevisiae GriP mutaseresidues, S55 and H181, form a strong (0.26 nm) hydrogenbond (Fig. 1). The loss of this hydrogen bond in the S62Amutant could perturb the active-site architecture. The tertiaryand quaternary structures of dGriP mutases seem to be sensitiveeven to small active-site changes. For example, the S. cerevisiaeS11A mutant showed a series of structural abnormalities [8].The S. cerevisiae H181A mutant was also shown to be lessstable than wild-type enzyme, particularly in the presence ofGri2,3P2 [10].

Spectroscopic properties of wild-type and mutant GriPmutases

CD spectra. The far-UV CD spectra of wild-type GriP mutaseand the various mutants studied were found to be generally verysimilar; representative data for the R14Q, E93D, and R120Qmutants are shown in Fig. 3a. When analysed over thewavelength range 240±195 nm by the selcon and varslcprocedures [29], the secondary-structure content of the wild-type GriP mutase was found to be 31% a helix, 20% b sheet,19% b turns and 30% remainder. The values for a helix and bsheet compare favourably with those determined by NMR, i.e.34% and 22%, respectively [19]. Application of the continprocedure [29] yielded 34% a helix and 32% b sheet; possiblereasons for the overestimation of the b-sheet content have beendiscussed previously [30]. The spectra of E93D and R120Q arevery similar to those of the wild-type, whereas that of R14Qshows a significant decrease in molar ellipticity (23% reductionat 225 nm). Analysis of the spectrum of R14Q suggests that theb-sheet content is largely unchanged from wild-type GriPmutase, whereas the a-helix content is reduced to 26%.

The near-UV CD spectra of wild-type and the same selectedmutants are shown in Fig. 3b. The dominant features of thewild-type spectrum in this region are the inflection points at285, 289, 293 and 298 nm, which arise from the Trp sidechains in the protein as a result of exciton coupling [31].Although the overall features are similar in each spectrum,there are some differences in the sizes of the various peaks inthis region.

Taken together, the far-UV and near-UV CD data suggestthat, while the overall folding of the wild-type and mutantenzymes is likely to be similar, there may be some subtlechanges in the secondary and tertiary structures. In the case of

Fig. 4. 1H-15N HSQC NMR spectrum of the wild-type (wt) and E93D mutant of S. pombe GriP mutase. The spectra of wild-type and E93D mutant

enzymes are shown in panels (a) and (b), respectively. In the E93D mutant, small changes (labelled) were observed in the chemical shifts of the main-chain

amide groups of residues close to the site of mutation. The most significant change was observed for the N165 amide peak, which is thought to be due to

disruption of a hydrogen bond between N165 and the side chain of the carboxylate of residue 93, caused by the mutation from E to D.


the R14Q mutant, however, the structural changes are likely tobe significant. As noted above, this mutant shows a decreasein amplitude in the far-UV CD spectrum, and in the near-UVCD spectrum there are differences in the 295±300 nm and260±275 nm regions, characteristic of Trp and Phe, respec-tively. The structural perturbations could well be responsiblefor the altered stability of this mutant towards denaturation, asdiscussed in the next section.

Inspection of the structure of S. cerevisiae GriP mutaseshows that the equivalent residue R7, which is adjacent to thecatalytically important H8, also interacts with a number ofactive-site residues and thus may be important in maintainingthe correct orientation of these critical side chains [7,8]. As aconsequence, substitution of a glutamine for an arginine residueat this position may compromise the structure and stability ofthe enzyme.

The 1H-15N HSQC NMR studies on the E93D mutant [19]confirmed that its overall structure was very similar to that ofwild-type, although there were small changes in the environ-ment of a few main-chain amide groups, particularly G16, G28,E89, R94, H163, N165 and S166 (Fig. 4).

Fluorescence spectra. When excited at 290 nm, wild-type GriPmutase shows a fluorescence emission maximum at 336 nm,indicating that the four Trp side chains are on averagemoderately exposed to solvent [32]. In the case of themutant enzymes, the emission maxima were all within therange 335±338 nm, implying that the average environments ofthe Trp side chains (broadly reflecting the tertiary structures) inthe mutants are generally similar to those in the wild-type(Fig. 3c). However, the intensity of the emission spectrumof the R14Q mutant is significantly different from that of thewild-type and the other mutant enzymes, indicating thatstructural perturbations have occurred. The changes influorescence presumably reflect alterations to the environmentof one or more of the Trp side chains in this mutant, asindicated by the changes in the near-UV CD spectrum (Fig. 3b).

Stability of the folded state of wild-type and mutantenzymes

The unfolding of each protein by GdmCl was monitored by thechanges in ellipticity at 225 nm and in fluorescence emissionintensity at 350 nm. These parameters reflect the loss ofsecondary and tertiary structure of the protein, respectively.Typical unfolding curves are shown in Fig. 5a,b for the wild-type and R94Q mutant enzymes, respectively; the data have

been normalized to show the changes relative to the maximumchange occurring between 0 and 6 m GdmCl. For bothenzymes, it is clear that the changes in fluorescence and CDrun in parallel with each other and this was also observed forthe other mutants studied. In all cases, the unfolding datacould be analysed satisfactorily in terms of a simple two-statemodel, using linear extrapolation [24] (Fig. 5) in order to givethe stability parameters summarized in Table 1. The stabilityof the wild-type enzyme is towards the lower end of therange of typical stability values observed for globular proteins

Fig. 5. Unfolding of S. pombe GriP mutase by GdmCl. Panels (a) and (b)

show data for wild-type and R94Q mutant, respectively. Circles and squares

show the relative changes in ellipticity at 225 nm and fluorescence at

350 nm, respectively. The insets show the data analysed in terms of a two-

state model [24] with linear extrapolation.

Table 1. Stability data for S. pombe wild-type and mutant GriP mutase towards unfolding by GdmCl. The extent of unfolding for each enzyme was

obtained from the changes in fluorescence and ellipticity in the presence of increasing concentrations of GdmCl. The unfolding data were analysed according

to a two-state model with linear extrapolation [24]. In each case, the correlation coefficient for linear regression was 0.98 or greater. The figures in

parentheses represent one standard deviation of the estimate. [GdmCl]0.5 represents the concentration of denaturant required for 50% unfolding, and m is the

slope of the plot of DG against [GdmCl].

GriP mutase enzyme DG (kJ´mol21) [GdmCl]0.5 (m) m (kJ´mol21´m21)

Wild-type 23�.3 (0.9) 0�.68 (0.03) 234�.4 (1.3)

R14Q 12�.4 (0.9) 0�.98 (0.07) 212�.6 (1.1)

E93D 28�.3 (1.9) 0�.81 (0.06) 234�.8 (2.2)

R94Q 19�.0 (1.2) 0�.71 (0.05) 226�.8 (1.6)

R120Q 19�.9 (1.0) 0�.82 (0.04) 223�.1 (1.4)

R121Q 17�.4 (0.9) 0�.67 (0.04) 225�.9 (1.2)

R120,121Q 25�.4 (1.5) 0�.80 (0.05) 231�.9 (2.0)


(20±65 kJ´mol21) [33]. The E93D and R120,121Q mutants areslightly more stable than wild-type enzyme, and in the case ofthe former mutant, this stability is reflected in significantlylonger sample lifetimes during NMR studies [19]. The R94Q,R120Q and R121Q mutants are somewhat less stable. The mostsignificant change is observed in the case of the R14Q mutantfor which the stability is about 50% that of the wild-typeenzyme. It should be noted that, within the overall stability

values for the wild-type and mutant enzymes, there is noclear trend in the GdmCl concentration corresponding to50% unfolding or the slope (m) of the plots of DG vs. [GdmCl](Table 1). Although the detailed interpretation of theseparameters is a matter of debate [24], the data suggest thatthe mutations made may introduce variations in the interactionsstabilizing the tertiary structure and hence the degree of co-operativity of the unfolding process.

Table 2. Kinetic properties of S. pombe wild-type and mutant GriP mutases. The assays of mutase and phosphatase activities were performed as

previously described [9]. All mutants retained Gri2,3P2-dependence of mutase activity. Kinetic data for the mutase activity were fitted to the Michaelis±

Menten equation using the program leonora [23]. The standard deviations in estimation of the Km and Vmax values are shown in parentheses. The assays of

phosphatase activity were performed in duplicate with less than 10% difference between the two readings. 2PG, 2-Phosphoglycollate.

Mutase activity Phosphatase activity (mmol´min21´mg21)

Vmax

(mmol´min21´mg21)

Km GriP

(mm)

Km Gri2,3P2

(mm)

Baseline

activity

1 1 mm 2PG

Wild-type 210 (7) 580 (55) 4.6 (0.06) 0.06 0.22

R14Q 1.8 (0.05) 1540 (95) 59 (4) 0.002 0.03

E93D 0.7 (0.015) 1250 (80) 14 (0.2) 0.03 0.04

R94Q 2.3 (0.1) 7460 (510) 36 (2.3) 0.24 0.26

R120Q 124 (5) 2640 (210) 28 (1.2) 0.15 0.15

R121Q 25 (1.3) 4900 (480) 34 (2.2) 0.16 0.17

R120,121Q 130 (4) 5260 (500) 49 (1.6) 0.20 0.22

Fig. 6. ITC data for binding of GriP to S. pombe GriP mutase. Panels (a) and (b) show data for wild-type and E93D mutant, respectively. The calorimetry

data were analysed by nonlinear regression in terms of a simple single-site binding model using the Microcal origin software package.


Kinetic properties of the active-site mutants

The kinetic properties of the active-site mutants and wild-typeGriP mutase are summarized in Table 2. These properties canbe used to draw some conclusions about the importance of thevarious side chains in catalysis. However, it should be borne inmind that such conclusions rely on the assumption that nostructural changes have occurred in the mutants, and this isalmost certainly not the case in the R14Q mutant, as discussedabove. The mutase activity is drastically reduced relative to thewild-type value for three of the mutants, R14Q, E93D andR94Q, which exhibit 117-fold, 300-fold and 91-fold reductionsin Vmax, respectively. These values are consistent with thesuggestion that R14Q, E93D and R94Q are involved inthe catalytic mechanism. For R14Q and E93D, the Km valuesfor GriP were raised by a mere 2- to 3-fold, further suggestingthe importance of these residues in catalysis rather thansubstrate binding. However, in the case of R94Q, the Km forGriP is 13-fold higher than the wild-type value suggesting R94also plays a role in substrate binding. Indeed, R94Q alsoexhibits a much higher Km for Gri2,3P2 (eightfold higher thanwild-type), indicating the importance of this residue in bindingmonophosphoglycerates and bisphosphoglycerates. The mutantexhibiting the highest Km value for Gri2,3P2 was R14Q, inagreement with a possible role for this residue in binding thethe carboxylate group of Gri2,3P2. It has been recognized that,in view of the multistep ping-pong mechanism of the mutasereaction and the very rapid phospho-transfer steps involved, itmay be misleading to interpret changes in Km values solely interms of changes in substrate-binding affinities [34,35]. Indeed,direct determination of the enzyme±GriP dissociation constantby (ITC) gives a value for Kd much smaller than the Km valueobtained under similar conditions, as described in the next section.

Compared with the wild-type enzyme, the phosphataseactivity is reduced for the R14Q and E93D mutants. Itshould be noted, however, that the decrease in phosphataseactivity is less than the decrease in mutase activity. Thephosphatase activity of the R14Q mutant is enhanced in thepresence of 2-phosphoglycollate as is that of the wild-typeenzyme. The reduction in phosphatase activity could be a directresult of poor phospho transfer. The R94Q mutant, despite thehigh Km for Gri2,3P2, has an increased phosphatase activitywhich is unaffected by the presence of 2-phosphoglycollate. AsR94Q appears to be poor at binding monophosphoglyceratesand bisphosphoglycerates, it is possible that water may gainaccess to the active site more readily, thus promoting dephos-phorylation of the enzyme, which is the distinct step in thephosphatase activity. It is likely that 2-phosphoglycollatemimics the binding of the substrate, and its lack of effect onthe phosphatase activity of the R94Q mutant could reflect muchweaker binding of this ligand.

The Km values for GriP and Gri2,3P2 of the mutants R120Q,R121Q and R120,121Q suggest the importance of theseresidues in the binding of monophosphoglycerate and bisphos-phoglycerate substrates. The Vmax values for the mutaseactivities of the R120Q and R120,121Q mutants are relativelyunaffected and are more than 50% of the value for wild-type enzyme; that for the R121Q mutant is some 12% of wild-type. These reductions are in marked contrast with the drastic,100-fold or greater, reductions observed in the R14Q, E93D andR94Q mutants.

The phosphatase activity is enhanced as a result of mutatingresidues 120 and/or 121. Again, this enhanced activity could beexplained by the poor binding of monophosphoglycerates andbisphosphosphoglycerates, allowing water to gain access to the

active site thereby promoting the phosphatase activity.Similarly, the inability of 2-phosphoglycollate to stimulate thephosphatase activity of R120Q,R121Q or R120,121Q could bedue to poor binding of this ligand.

Substrate-binding studies

ITC was used to examine the binding of GriP to wild-typeenzyme, E93D (a `phospho-transfer' mutant) and R120,121Q(a `substrate-binding' mutant). The results are shown in Fig. 6.The binding of GriP to the nonphosphorylated form of GriPmutase resulted in a nonproductive complex and permittedmeasurement of the enthalpy change on binding GriP. (In thecase of Gri2,3P2 binding, there could well be complicationscaused by the enthalpy of the phospho-transfer reaction.) Thebinding of GriP to wild-type GriP mutase was a mildlyexothermic process. After correction for the heat effects ofdilution, the titration data were found to be consistent with tight1 : 1 enzyme±substrate complex formation, with an apparentassociation constant Ka of 2.5 � 105 m21 (corresponding to aKd of 4 mm) and a DH � 26.0 kJ´mol21. However, the bindingof GriP to the E93D mutant was mildly endothermic; analysisof the data suggested rather weaker binding (apparentKa � 3.1 � 104 m21, corresponding to a Kd of 32 mm;DH � 4.6 kJ´mol21). The ITC results for the R120,121Qmutant were inconclusive, with little or no apparent heatchange on the addition of GriP, indicative of either very weakbinding or no detectable binding under the conditions studied.These results strongly suggest a different binding modefrom that previously observed crystallographically for theS. cerevisiae enzyme [11]. In that binding mode, no GriP atomis closer than about 0.78 nm to either R113 or R114. InsteadR113 and R114 bind one of two sulfate ions. That sulfate issimilarly positioned to one of the two previously observed in adifferent crystal form [8]. It seems that the high sulfateconditions used to grow the GriP-complexed crystals [11] led tocompetition for binding in the active site between sulfate ionsand GriP. The second sulfate ion, binding in the same place asa phospho group of GriP, reduced the GriP occupancy to 0.4[11]. The binding results for the S. pombe R120,121Q mutantreported here suggest that competition from the first sulfate ionmay have prevented GriP from binding in its physiologicallyrelevant mode.

It should be noted that, in the case of the wild-type enzymeand E93D mutant, the relatively small enthalpy changesobserved on binding GriP must imply that the reactions arelargely entropy-driven, presumably as the result of release ofa substantial amount of bound water on complex formation.For binding of GriP to the wild-type and E93D mutants,the DS values at 25 8C are calculated to be 183 and1101 J´mol21´K21, respectively. This conclusion is consistentwith the presence in the active site of subunit A of the 0.21-nmcrystal structure of S. cerevisiae dGriP mutase of a well-ordered network of 18 water molecules as well as two sulfateions [8]. The Kd values for the wild-type enzyme and the E93Dmutant are both much smaller than the observed Km values forthese substrates, illustrating the difficulties referred to in theprevious section in interpreting the kinetic parameters in termsof affinities.

MS analysis of wild-type and mutant enzymes

Mass spectra of the wild-type and mutant enzymes gave massesthat were in accordance with the amino-acid sequences. Usingthe procedures described in previous studies [36], we were ableto observe the formation of the phosphoenzyme intermediate of


the wild-type and all the mutant enzymes. Phosphorylation wasmonitored by an increase in mass of 78 Da following theaddition of Gri2,3P2. The t1

2values of the phosphorylated form

of the various enzymes were deduced, with wild-type, R14Q,R94Q, R120Q, R121Q and R120,121Q all showing a t1

2of less

than 1 min, as noted in earlier work [36]. However, in the caseof the E93D mutant, the phosphorylated form was more stable,with a t1

2of 10 min, indicating that E93 plays an important role

in phospho-transfer reactions from the catalytically importantH15 side chain.

Implications for the mechanism of GriP mutase and relatedenzymes

Recent studies on S. cerevisiae GriP mutase [8] and thestructurally homologous rat testis Fru2,6P2ase [12] have ledto a proposed mechanism of enzyme phosphorylation. In thepresent work, site-directed mutagenesis of S. pombe GriPmutase was used to test the functional importance of a numberof active-site residues (R14, S62, E93, R94, R120 and R121)implicated in this type of mechanism. Sequence alignments[37] and structure-based modelling studies indicate that R14,S62 and E93 are conserved in all dGriP mutases andFru2,6P2ases, further suggesting a phospho-transfer role forthese active-site residues. However, R94, R120 and R121 areunique to the dGriP mutase sequences, which lends support totheir putative role in substrate binding.

In general, the kinetic data reported in this paper areconsistent with a catalytic role for R14 and E93 and a bindingrole for R120 and R121, although these conclusions should betreated with caution in view of possible localized structuralalterations as a result of the mutations. The much reduced Vmax

value together with the large increase in Km values for R94Qsuggest a dual role for R94. In the case of R14Q, some cautionmust be exercised in interpreting the results because of thesubtle structural changes that have occurred in the mutant;however, R14 would appear to be of importance in the catalyticcycle. Modelling studies [8] suggest that either of theequivalent S. cerevisiae GriP mutase residues R7 or K97would be suitably positioned to interact with the carboxylategroup of Gri2,3P2. Previously, K97 was favoured [8], but thedramatic effects of the R14Q mutation on the properties ofS. pombe GriP mutase suggest that this may indeed be the roleof R7 (R14 in the S. pombe enzyme). Figure 1 shows acorresponding model of Gri2,3P2 bound to the S. cerevisiaeGriP mutase active site with the carboxylate moiety forminga 0.28-nm ionic interaction with Arg7. An equally favourableGri2,3P2-binding mode, retaining the carboxylate±R7 inter-action, can be obtained by swapping the positions of the twophospho groups. Furthermore, modelling suggests that thereorientation of Gri2,3P2 can be accomplished without lossof the carboxylate±R7 interaction. Hence a `tethering' role forR7 during reorientation of Gri2,3P2 seems possible. TheGri2,3P2-binding mode in Fig. 1 is not related to the bindingmode reported crystallographically for GriP [11] but, asmentioned above, the mutant data reported here and otherconsiderations suggest that that GriP-binding mode may not bethe physiologically relevant one.

In the mutase assay, the E93D mutant was the least active ofthose studied (300-fold less active than wild-type) with only atwofold to threefold increase in Km values. Mutations of theequivalent glutamate residue in S. cerevisiae GriP mutase (E86)[8] and the structurally related Fru2,6P2ase (E327) [14] resultedin a loss of activity: the S. cerevisiae E86Q mutant retained lessthan 1% of the wild-type GriP mutase activity and E327A,

E327Q and E327D retained 4, 2 and 20% of the wild-typeFru2,6P2ase activity. These findings led to the proposal of anacid catalyst role for this glutamate residue. The kinetic datatogether with the phosphoenzyme stability studies of S. pombeGriP mutase E93D support this proposal. However, theconservative substitution from a glutamate to an aspartateresidue at this position had a much more pronounced effect onthe S. pombe GriP mutase than on the rat liver Fru2,6P2ase.This difference may reflect spatial differences in the active-siteresidues of these homologous enzymes.

Of the mutants studied, only R94Q appeared to showsignificant changes in both substrate/product/cofactor bindingand catalysis. This mutant displayed a 91-fold loss ofactivity with a concomitant increase in Km for GriP andGri2,3P2 (13-fold and eightfold higher than wild-type,respectively). These findings support both the S. cerevisiaeGriP mutase modelling studies [8] and S. pombe GriP mutaseNMR titration studies (S. Uhrinova, P. N. Barlow, L. A.Fothergill-Gilmore, J. Nairn & N. C. Price, unpublished work)which have suggested the importance of R94 in binding bothmonophosphoglycerates and bisphosphoglycerates and in thecatalytic cycle. The decreased Vmax seen for the R94Q mutantmay constitute the first experimental evidence for the proposedreorientation of Gri2,3P2 as part of the reaction mechanism ofdGriP mutase [1]. Crystal structures of S. cerevisiae dGriPmutase revealed sufficient space for this reorientation to occurpartly because of the absolute conservation of a glycine atposition 21 [8]. Modelling of the reorientation showed R87 andR59 to be well placed to interact with phospho groups ofGri2,3P2 during the reorientation, a hypothesis supported by thekinetic properties of the S. pombe R94Q mutant.

The kinetic properties of S. pombe GriP mutase mutantsR120Q, R121Q and R120,121Q indicate a possible substrate-binding role for these residues. Indeed, the equivalent residuesin S. cerevisiae GriP mutase (R113 and R114) are positionedon the surface loop (92±145), and modelling studies haveimplied their importance in hydrogen bonding with Gri2,3P2.Likewise the basic residues (R352, K356 and R360) on thesurface loop (331±362) of Fru2,6P2ase have been shown to beimportant in substrate binding [38].

In summary, therefore the work described here confirms andextends the proposals made about the roles of various sidechains in the mechanism of dGriP mutase. The continuingNMR studies will provide further structural and dynamicinformation about the overall catalytic cycle.

A C K N O W L E D G E M E N T S

This work was supported by the Wellcome Trust. The CD and the

Biological Microcalorimetry facilities are supported by the Biotechnology

and Biological Sciences Research Council (BBSRC) and Engineering and

Physical Sciences Research Council (EPSRC). The mass spectrometer

and NMR facilities are supported by the BBSRC and the Office of Science

and Technology through the Edinburgh Centre for Protein Technology. We

wish to thank Margaret Nutley and Professor Alan Cooper for obtaining and

interpreting the ITC data, Dr Adam M. Gouldsworthy and Sally Shirran for

processing MS data, and Elaine Small and Craig Laurie for preliminary

studies on the R114Q and R113,114Q mutants, respectively.

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Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase

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