PROTEINS: Structure, Function, and Genetics 10:315-324 (1991)

Conformational Changes in Yeast Phosphoglycerate Kinase Upon Ligand Binding: Fluorescence of a Linked Probe and Chemical Reactivity of Genetically Introduced Cysteinyl Residues Michel Desmadril,' Philippe Minard,' Nathalie Ballery,' Sophie Gaillard-Miran,' Len Hall: and Jeannine M. Yon' 'Laboratoire d'Enzymologie physico-chimique et molCculuire, Groupe de Recherche du Centre National de la Recherche Scientifique assock?a 1'UniversitC de Paris-Sud, 91405 F, Orsay, France; and 'Department of Biochemistry, University of Bristol, School of Medical Sciences, Bristol BS8 1 TD, U X .

ABSTRACT The effects of ligands on the conformation of yeast phosphoglyceratekinase were explored by introducing cysteinyl residues at different positions in the molecule by site-directed mutagenesis. Thus several mutants were constructed, each containing a unique cysteinyl residue. Neither the conformation nor the enzyme activity was affected by the substitutions. The reactivity of the thiol groups and the fluorescence of N-acetyl-Nf-(5-sulfo1-naphty1)ethylene-diamine covalently linked to these thiols were used to monitor the conformational changes induced upon ligand binding. It was found that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment of residues 35 and 183. No alteration was observed on the external side of the protein. Only 3-Phosphoglycerate induced these conformational changes. However, when the fluorescent probe was attached to residue 377, the binding of the two substrates was required to induce a modification in the fluorescence of the probe. These results indicate that the substrates separately or together induce discrete molecular motions in phosphoglycerate kinase.

tional properties of the enzyme. The structural features have suggested that a large conformational change might take place during the catalytic cycle. The large distance between the nucleotide binding site and the site proposed for phosphoglycerate substrates has allowed Banks et aL6 to propose the existence of a hinge bending motion that would bring the two substrates close together in a water-free microenvironment favorable for catalysis. Several different experimental results have supported this hypothesis: (1) small-angle X-ray scattering studies have shown a small but significant decrease of the radius of gyration for yeast' and pig muscleg phosphoglycerate kinase upon simultaneous binding of 3-phosphoglycerate and Mg-ATP, (2) nuclear magnetic resonance studies of yeast phosphoglycerate kinase show a number of small substrate-induced conformational effects both close to and remote from the 3-phosphoglycerate binding site;lo9l1 (3) significant conformational changes induced by the substrates have been probed by variation in the reactivity of the single thiol group of the yeast protein;12 furthermore, 3-phosphoglycerate decreased the reactivity of the five slowly reacting thiol groups of the rabbit13 and pig muscle14 enzymes. The use of site directed mutagenesis to investigate the transition between the two hypothetical "open"

Key words: site-directed mutagenesis, cysteine, phosphoglycerate kinase, IAEDANS INTRODUCTION Yeast phosphoglycerate kinase is a monomeric enzyme with a molecular mass of 44500 Da,' which catalyzes the reversible transfer of a phosphoryl group from 1,3-bisphosphoglycerateto ADP, in the first ATP producing step of glycolysis.2 The threedimensional structures of the yeast3s4 and horse enzymes are very similar. The most characteristic feature of the molecule is the presence of two well-resolved domains of approximately equal size, separated by a deep cleft. Several motions seem to play a role in the func0 1991 WILEY-LISS,INC.

Received August 7, 1990; revision accepted December 18, 1990. Address reprint requests to Dr. Jeannine M. Yon, Laboratoire d'Emymologie physico-chimique et moleculaire, Groupe de Recherche du Centre National de la Recherche Scientifique, Universit6. de Paris-Sud, CNRS batiment 430,91405 F, Orsay Cedex, France. Abbreviations: 3PG, 3-phosphoglycerate; ADP, adenosine 5'diphosphate; AEDANS, N-acetyl-N'-(5-sulfo-l-naphtyl)ethylene-diamine; ANS, 8-Anilino-1-naphtalenesulfonicacid; ATP, adenosine 5'-triphosphate; CD, circular dichroism; EDTA, ethylenediaminetetraacetic acid; IAEDANS, N-Iodoacetyl-N'-(5-sulfo-l-naphtyl)ethylene-diamine;Nbs,, 53'Dithiobis-(2-nitrobenzoic acid); NMR, nuclear magnetic resonance; NTCB, 2-Nitro-5-thiocyanobemoic acid; PGK, phosphoglycerate kinase (E.C. 2.7.2.3); Tris, tris[hydroxymethyll-aminomethane.

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and "closed' forms has been attempted on the yeast enzyme. These experiments designed to test the hypothesis t h a t a trigger mechanism exists involving a salt bridge between histidine 388 and glutamate 190,'" have included several substitutions at both Ifi. 17 positions. In another study, Arg 168, thought to be involved in stabilizing the transition state intermediate, was substituted by Lys or Met.'" These different mutations have been found to significantly affect the enzyme activity. In all of these studies, although the observed changes in the structural properties or enzyme activity could reflect the existence of a domain movement, they do not clearly indicate if such changes in the enzyme's structure a r e identical to those assumed from the crystallographic studies.'" In the present work, the conformational changes in phosphoglycerate kinase upon ligand binding were reinvestigated by following the variation in properties of local probes. The chemical reactivity of a side-chain can be considerably pertubed by local molecular rearrangements. Therefore, the study of known and reactive side-chains such as thiol groups can provide valuable information on the nature and mechanism of the conformational changes observed in phosphoglycerate kinase. Fluorescence spectroscopy of fluorescent probes covalently linked to the protein is another valuable approach to monitor conformational changes. However, both methodologies are clearly limited to situations in which a suitable reactive side-chain can be specifically used for chemical modification. An excellent way to introduce such residues is now provided by site-directed mutagenesis. In this work, cysteinyl residues were introduced at different positions in the protein. For this purpose, several mutant proteins were expressed, each containing only one internal cysteinyl residue at a defined position. The reactivity of the thiol group was directlylused to monitor the conformational changes induced upon ligand binding. The thiol groups were also used for t h e covalent attachment of a n extrinsic fluorescence probe: N-acetyl-N'-(5-sulfo-l-naphtyl)ethylene-diamine.

MATERIALS AND METHODS Mutagenesis Mutations were introduced by site-directed mutagenesis as described by Minard et a1." The following mutants were obtained: (Cys 97-+Ala, Ser 35Cys); (Cys 97+Ala, Ser 1 4 h C y s ) ; (Cys 97+Ala, ; Ala 1 8 3 - 4 ~ s ) ;(Cys 97-Ala, Glu 2 4 9 4 ~ s ) (Cys 97--.Ala, Ala 3 7 7 j C y s ) . They are referred to as C97A,S35C; C97A,S140C; C97A,A183C; C97A, E249C and C97A,A377C. The yeast strain used in these studies also expressed a low level (less than 10% of wild-type chromosomal gene phosphoglycerate kinase (PGK, E.C. 2.7.2.3.).

Enzyme Purification Each protein was purified as described by Minard et al.'l

Enzyme Activity Measurements Enzyme activity was measured as described by Bucher'" using a coupled assay with glyceraldehyde-3-phosphate dehydrogenase. The protein concentration was determined using a n absorption coefficient, A'rkl,, = 4.9 at 279 nrn.':'

Reactivity of Thiols Towards 5,5'-Dithiobis-(2-Nitrobenzoic Acid) and 2-Nitro-5-Thiocyanobenzoic Acid All experiments were carried out at 20°C in a 100 mM Tris-HC1 buffer pH 7.5, containing 500 pM EDTA either in the absence or the presence of substrates. To follow the kinetics of thiol reactivity toward Nbs,, the reaction was started by the addition of 10 pl of a 10 mM reagent stock solution to 1 ml of a 2 pM PGK solution. The variation in absorbance at 412 nm (E = 14150 M c m - ' 24) was recorded with a CARY 219 spectrophotometer equipped with a thermostatted cell holder and connected to a microcomputer for data acquisition. The reactivity of thiol toward NTCB was studied under the same conditions, using the same absorbance coefficient.'" For each individual experiment, the apparent first-order rate constant was calculated from the whole set of data by a nonlinear regression method (Newton-Raphson algorithm).'6 The second-order rate constant was calculated taking into account the concentration of the reagent (Nbs2 or NTCB). The low level of wild-type PGK did not interfer with the chemical modification of the mutants since its reactivity is two orders of magnitude lower than the slowest reacting cysteinyl residue (183),as previously ~ h o w n . ' ~

Fluorescent Labeling Fluorescent labeling of each mutant protein at the single cysteinyl residue was achieved by incubating the protein at 20°C in the dark in a 100 mM TrisHC1 buffer pH 7.5 containing 500 pM EDTA and IAEDANS (Sigma). The final concentration of IAEDANS was calculated to obtain a cysteine to reagent ratio of 1:20. The incubation time was adjusted to allow complete labeling (2 to 12 h depending on the mutant). The labeled protein was then desalted on a small Sephadex' G25 column (15 x 1 cm) in a 100 mM Tris-HCI buffer pH 7.5, and the degree of labeling was determined from the absorption spectrum of the labeled protein using a E for IAEDANS of 6,100 M-'-cm ' at 350 nm;2x the protein concentration being determined by Fohn titration." Under these experimental conditions, wild-type PGK was not labeled.

LIGAND EFFECT ON YEAST-PGK

Fluorescence Measurements Fluorescence data were obtained using a 10 mm cuvette at 23°C in a Perkin-Elmer spectrofluorimeter model MPF 44B, equipped with a xenon lamp, a thermostatted cell holder, and connected to a microcomputer for data acquisition. Emission fluorescence spectra were recorded with a slit of 3 nm for both excitation and emission, the excitation being performed a t 350 nm. Ligand binding was determined either directly with AEDANS fluorescence or indirectly with ANS fluorescence. The titrations with substrates, of the labeled enzyme solution or ANSenzyme solution, were made as addition tit ration^.^' To 2.5 ml of a labeled PGK solution or a PGK-ANS solution (at a concentration of 20 and 7 pM, respectively), small volumes of ligand were added with a micropipette (100 p1 total), the fluorescence intensity being corrected by the dilution factor. To determine the dissociation constants of the ligands to the labeled protein, the fluorescence intensity changes were analyzed using the following equation:

where F is the fluorescence intensity in the presence of ligand a t the concentration S, F, the fluorescence intensity in the absence of ligand, F the fluorescence intensity in the presence of ligand a t saturating concentration and K, the dissociation constant. For K, measurement of Mg-ATP, performed in the presence of ANS, the following equation was used: F - Fo = (F, - Fo)

bl+S

+

=]

(11)

&2+S

where the parameter (Y represents the proportion of the first saturation curve and Kal, Kd2 the two apparent dissociation constants.

RESULTS Choice of the Mutation Positions and Effect on the Enzyme Structure Since wild-type yeast phosphoglycerate kinase already contains one buried cysteinyl residue (Cys 97), a first mutant devoid of cysteine was constructed (C97A). Then, from this first mutant, several others were obtained by further rounds of mutagenesis, each containing one internal cysteinyl residue a t differing positions. The positions where each cysteinyl residue was introduced were chosen to be located in the cleft between the two domains (positions 35, 183, and 377) or, as a control, on the external side of each domain (positions 140 and 249), as shown in Figure 1. To minimize possible deleterious effects caused by the mutations, the cysteinyl residues were introduced in weakly conserved positions. The nature of the aminoacid present at these positions in

317

Fig. 1. Schematic drawing of yeast PGK. The square at position 97 indicates the location of the wild-type cysteinyl residue which was replaced by an alanine in all mutants. The circles indicate the positionswhere the cysteinyl residues were introduced.

other species and their relative accessibility were also used as criteria (Table I).

Effect of Ligands and Ions on SH Group Reactivity In the presence of a large excess of Nbs2, the modification of the unique thiol group of each mutant protein followed monophasic kinetics. However, depending on the location of the cysteinyl residue, the rate constant varied between 80 and 3.104 M-lsec-l. Under the same experimental conditions, for protein C97A,S35C, the rate constant of the kinetics with Nbs, was so high that it was only possible, by manual mixing, to evaluate a lower limit value (3.104M-lsec-l). For this mutant protein, the effect of ligands on thiol group reactivity was studied using NTCB. With this reagent, the second-order rate constant was 350 M-'.sec-'. Despite the huge variation in the intrinsic reactivity of each thiol group, the cysteinyl residues located in the cleft (C97A,S35C, C97A,A183C, and C97A,A377C), displayed the same general features with respect to the effect of ligands (Table 11).3PG, as well as nucleotides, reduced the apparent firstorder rate constant, the effect being very much smaller for the mutant protein C97A,S35C, MgADP was found to have a very strong protective effect on mutant proteins C97A,A183C and C97A,A377C. Generally, both Mg-ATP and 3PG reduced the reactivity to a lower extent, except €or C97A,A183C, where the 3PG protective effect was similar to that of Mg-ADP. For mutant protein C97A,A377C, the high efficiency of Mg-ADP in protecting the thiol group is clearly related to the position of the metal ion in the Mg-ADP complex,21 whereas for C97A,A183C, ADP and Mg-ADP had the same protective effect. For mutant protein C97A,S35C, the protective effect of ligands was less pronounced. However, it was not a secondary effect due to the use of NTCB in-

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TABLE I. Specific Activity and Characteristics of the Side Chain Substitution of Wild-Type and Mutant PGK ~

PGK Wild type PGK C97A,S35C C97A,S140C C97A,A183C C97A,A249C C97A,A377C

Specific activity (Wmn) 2100 1700 1500 2100 2400 2150

Residue at the same position in other species* CCCCCCY NNNNNNS EEEAADE GGGAASA EEESSKD CCCAAAA

*Order of the different species: Horse, Human, Mouse, Trypanosoma brucei B, Trypanosoma brucei C, Aspergillus nidulans, Escherichia coli.

stead of Nbs, for monitoring the accessibility of the thiol group. Indeed, using Nbs2,no significant effect on the thiol group reactivity upon ligand binding was observed. For mutant proteins C97A,S140C, and C97A, 249C whose cysteinyl residues are located far away from the cleft, the ligand binding did not affect the thiol group reactivity, whereas for mutant C97A, S140C the effect of ligand binding had no significant effect and for mutant C97A,E249C, it resulted in a slight increase in the thiol group reactivity. Since it has been shown that anions may have a pronounced effect on substrate binding,23kinetics of enzyme activity,31 and the effect of phosphate and sulfate ions was also studied. For mutant protein C97A,A183C, these two anions displayed a strong protective effect, comparable to that of MgADP. On protein mutant C97A,S140C, whose cysteinyl residue is far away from the cleft, these anions did not affect the reactivity of the thiol group. Effect of SH Labeling Upon Enzyme Reactivity The residual activity of each mutant protein was measured after modification of the thiol group by Nbs,. For those mutant proteins whose thiol group is located in the cleft (C97A,S35C, C97A,A183C, and C97A,A377C), the residual activity was always less than 20%. This low level of activity was probably partially due to the small fraction of wild-type PGK present in these preparations.'l In contrast, mutant proteins C97A,S140C and C97A,249C retained, respectively, 75% and 100% of their enzyme activity after modification by Nbs, (Table 111).

Fluorescence Properties of AEDANS Labeled Proteins The effect of ligand binding upon fluorescence properties of AEDANS was analyzed using five mutant proteins: C97A,S35C, C97A,S140C, C97A, A183C, C97A,249C, and C97A,A377C. The results appeared to be very dependent upon the probe's lo-

cation and the substrate bound to the protein (Table IV).

Effect of 3PG binding 3PG binding induced changes in the fluorescence properties of the AEDANS only in C97A,S35C and C97A,A183C mutant proteins. For mutant protein C97A,S35C, 3PG binding induced a fourfold increase in the fluorescence emission, accompanied by a 15 nm blue shift of the maximum wavelength (Fig. 2A). For mutant protein C97A,A183C, the situation was totally different, since 3PG binding induced a 20% decrease in the fluorescence emission, with a 5 nm red shift of the maximum wavelength (Fig. 2B). For mutant proteins C97A,A377C, C97A,S140C, and C97A,249C, the binding of 3PG did not significantly modify the fluorescence properties of the probe (Fig. 2C,D). Effect of ATP binding For the five mutant proteins tested, Mg-ATP binding did not modify significantly the fluorescence properties of the probe (Fig. 2). Fluorescence properties of the ternary complex 3PG-ATP-PGK For mutant proteins C97A,S35C and C97A, A183C, the ternary complex possessed the same properties as the binary complex 3PG-PGK. In other words, the nucleotide did not modify AEDANS fluorescence emission on binding either to ligand free PGK or to 3PG-PGK binary complex (Fig. 2A,B). The mutant protein C97A,A377C appeared to give results differing from the other mutants. In fact, whereas the two binary complexes PGK-3PG or PGK-Mg-ATP did not lead to any change in the fluorescence properties of the probe, the ternary complex had a fluorescence intensity emission lowered to 50% with respect to the ligand-free protein, without any change in the maximum wavelength of the emission spectrum. Obviously, in this case there is a synergistic effect upon the binding of the two ligands. Moreover, this effect was not dependent on the order of substrate addition. For mutant proteins C97A,S140C and C97A, 249C, in the presence of ligands, the fluorescence properties were the same as those of the free-labeled enzyme: substrate binding did not modify the fluorescence properties of the probe. Determination of Substrate Binding Constants for Free and Labeled PGK The dependency of the fluorescence properties of AEDANS-PGK upon ligand binding was used to determine the binding constant of 3PG for labeled mutant proteins. The results were compared to the binding constants of the unlabeled mutant proteins and wild-type PGK. To determine the binding constants of the ligands

319

LIGAND EFFECT ON YEAST-PGK

TABLE 11. Relative Reactivity of Thiol Group in the Presence of Ligands*

Mutant No ligand (M-'.sec-l) 3PG ATP-Mg ADP-Mg ATP ADP Phosphate Sulfate

35t 3.104 80% 60% 60%

nd§ nd nd nd

140 300 100% 100% 100% nd nd 100% 100%

183 80 2% 20% 3% 15% 3% 2% 2%

249 950 94% 110% 125% nd nd nd nd

377% 370 14% 12% 4% 11%

18% nd nd

*The reactivity of thiol groups was measured under the conditions presented in Materials and Methods. To compare the effect of the different ligands on each mutant, the reactivities are reported relative to the intrinsic reactivity of the thiol group in the absence of ligand. tFor mutant protein C97A,S35C, the absolute rate constant corresponds to the lower limit value obtained using Nbs,. The relative values correspond to the results obtained using NTCB (absolute rate constant: 350 M-'.sec-'). $From Ref. 21. Bnd = not determined.

Table 111. Specific Activity of Mutant Proteins After Labeling With Nbsz* NbS,

C97A,V35C C97A,S140C C97A,A183C C97A,E249C C97A.A377C

labeled protein 20% 75% 10% 100% 10%

Cyanylated protein 92% ndt 80% nd t 71%

*Specific activity values are relative to the unlabeled mutant enzyme. tnd = not determined.

to the unlabeled mutant proteins, we used the procedure described by Wiksell and LarssonRaznikiewicz?, monitoring the modification of ANS fluorescence upon ligand binding. All the mutant proteins displayed the same features as the wildtype protein as described by Wiksell and LarssonRaznikiewicz.,, 3PG binding to an ANS-protein solution caused a large increase in the fluorescence intensity of the probe, without any change in the maximum wavelength. Whereas the binding of MgATP alone slightly decreased the fluorescence intensity of ANS, the addition of Mg-ATP to a solution of ANS 3PG-PGK led to a large decrease in the fluorescence intensity. These features were used to monitor the binding of each ligand. K,, was obtained as described in Materials and Methods, using the fluorescence enhancement of ANS upon 3PG binding (Fig. 3). For K, the signal used was the fluorescence decrease, induced upon nucleotide binding, of a n ANS-PGK solution containing 10 mM 3PG. In this case, the saturation curve was biphasic (Fig. 4). Therefore, equation I1 was used to analyze the experimental data. This equation corresponds to the existence of two independent binding sites. In fact, the existence of two binding sites for the nucleoti!l!8,@ already been proposed by several the low affinity site probably correauthors, sponding to nucleotide binding to the basic patch in

the N-terminal domain." The data given in Table 5 correspond to the K, value of the high affinity site. For the labeled mutant proteins C97A,S35C and C97A,A183C, whose fluorescence was dependent on 3PG binding, Kd,, was directly obtained by monitoring the variation of AEDANS fluorescence. The binding curve was analyzed using equation I (Fig. 3). For these proteins, Mg-ATP did not induce any change in the fluorescence properties of AEDANS. Thus the binding constant of this ligand was determined following the decrease in ANS fluorescence previously enhanced upon 3PG binding (10 mM 3PG). As for free PGK, biphasic saturation curves were obtained and equation I1 was used to analyze the data (Fig. 4). For the protein mutant C97A,A377C, all binary complexes did not lead to significant change in AEDANS fluorescence. Consequently the K, value for each ligand was determined by the variation in AEDANS fluorescence of a labeled protein solution containing the other ligand at saturating concentration.

DISCUSSION Previous studies have shown that the introduction of a unique cysteinyl residue in each mutant can 27 yield information on local events in a The studies presented in this work used this methodology to investigate in detail the conformational changes induced upon ligand binding to yeast PGK. The criteria used in choosing the positions to be mutated without change in site structure (i.e., the weak degree of conservation of the amino acid at this position and the nature of the amino acid at the same position in all PGK sequences) seem relevant. Indeed, far ultraviolet circular dichroism spectra (data not shown) of the different mutant enzymes were found to be indistinguishable from that of the wild-type spectrum, suggesting that regular secondary structures were not pertubed by the mutations. Moreover, the mutant PGKs retained enzyme activ-

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M. DESMADRIL ET AL.

Table IV. Ligand Binding Effect on the Spectral Characteristics of the Labeled Proteins*

C97A,S35C C97A,S140C C97A,A183C C97A,A377C

Free protein A Max nm 480 465 475 465

Relative fluorescence 400% 100% 80% 100%

+ 3PG

+Mg-ATP

+ 3PG A Max

nm 465 465 480 465

Relative fluorescence 100% 100% 100% 100%

+ Mg-ATP Max

A

nm 480 465 475 465

Relative fluorescence 400% 100% 80% 50%

A

Max nm 465 465 480 465

*The spectra were recorded as described in Materials and Methods, in the presence of saturating ligand concentration, i.e., 5 mM Mg-ATP or/and 5 m M 3PG. B

A m

m

3

a v

a, 0 (I

a, U

n

v) (u

L 0

-L 3

Wavelength

(nm)

I0

cl

Wavelength

(nm)

Wavelength

(nm)

12

,.

I1

3

a v a, U

c a, 0 v)

a, I 0 3

-LL Wavelength

(nm)

Fig. 2. Fluorescence emission spectra of C97A,V35C (A), C97A,S14OC (B), C97A,A183C (C), and C97A,A377C (D), without ligand ( l ) ,in the presence of 5 mM 3PG (2), 5 mM Mg-ATP (3) and in the presence of 5 mM 3PG and 5 mM Mg-ATP (4).

ity, most of them having the same specific activity as the wild-type enzyme. Only two mutant proteins had their specific activity slightly lowered. For mutant protein C97A,S140C, the specific activity was reduced to 75%,whereas protein mutant C97A,S35C retained 85%of the wild-type specific activity (Table I). These mutants had the same properties as the wild-type enzyme and thus closely reflected the molecular events occurring during the catalytic cycle. This was confirmed by the direct measurement of the dissociation constants of ligands bound to the mutant proteins: the values obtained (Table V)were

similar to those obtained for wild type PGK using different methods.23,33 The reactivities of the unique cysteinyl residues in the different mutant proteins depended upon their position in the structure, the second-orderrate constants ranging from 80 to 3.104 M-' sec-l. These differences cannot be solely correlated to different accessible surface areas, since one of the cysteinyl residue, in protein mutant C97A,S35C, had a reactivity higher than that of a free cysteine. Thus these variations in thiol reactivities must result from the local environment (hydrophilicity, polarity. . . ). For

321

LIGAND EFFECT ON YEAST-PGK

TABLE V. Dissociation Constants of Ligand for Free and AEDANS Labeled Mutant Proteins

‘BB 35

Wild type C97A,S35C C97A,A183C

Kd 3PG JLM Free Labeled protein protein 10 nd* 22 15

2 7

Kd Mg-ATP JLM Free Labeled protein protein 10 nd 10 4 6 2

*nd = not determined.

F

/

L3PGI

Fig. 3. Scatchard plot of 3PG binding to unlabeled ( 0 ) and labeled (0)C97A,V35C.

F

/

CMg-RTPI

Fig. 4. Scatchard plot of Mg-ATP binding to unlabeled ( 0 )and labeled (0) C97A,V35C.

this mutant protein, the local environment is mainly due to the neighboring amino acids in the sequence, since the same hyper reactivity was observed for the denatured protein. However, in spite of these huge differences in reactivity, all mutant proteins whose thiol group is located in the cleft (C97A,S35C, C97A,A183C, and C97A,A377C) had their reactivity affected on ligand binding. The effect was less pronounced for mutant C97A,S35C. This protective effect by the ligands is dependent on the thiol group’s location, since the thiol reactivity in mutant proteins C97A,S140C and C97A,E249C, located outside the cleft, did not significantly change upon ligand binding. The protective effect of the ligands could be simply explained by shielding of the SH groups. However, the distances between the SH groups located in different part of the cleft and the substrate binding sites are too long to allow a direct interaction between the substrates and the reagent. Furthermore, in the case of the strong protective effect of phosphate and sulfate ions, the size of these anions is not consistent with the shielding hypothesis. The change in reactivity of the cysteinyl residue in mu-

tant protein C97A,A377C was previously suggested to be the consequence of the ligand effect on the dynamic properties of helix XIL2l The maximum protective effect of the ligands was observed for protein mutant C97A,A183C, whose thiol group is located a t the bottom of the cleft. Nbs, labeling of the thiol groups located in the cleft (at positions 35, 183, and 377) led to a loss of enzyme activity, whereas mutant protein C97A, S140C retained 75% and mutant C97A,E249C was not affected (Table 111). Previous reports2l7 34 indicated a loss of enzyme activity after modification of the fast reacting thiols, this inhibition being dependent upon the size of the reagent, probably due to steric hindrance. The fact that Nbs, labeled proteins recovered more than 80% of activity after cyanylation, support this interpretation (Table 111). A very significant result obtained by these studies is that the steric hindrance leading to the loss of the activity was not due to the direct shielding of the ligand binding site by the reagent. In addition to the arguments given for Nbs, binding, this was also demonstrated by using AEDANS as a fluorescent probe. After labeling by IAEDANS, all mutant proteins, except C97A,S140C and C97A,E249C, displayed less than 10% of residual activity. However, these labeled proteins were able to bind 3PG since this substrate induced changes in the fluorescence properties of the probe. Moreover, using ANS as a probe, it was possible to monitor Mg-ATP binding to the AEDANS-labeled protein. These data indicate unambiguously that the probe did not prevent substrate binding while inactivating the enzyme. Another point to discuss is the significance of the observed changes in fluorescence properties of the probes upon ligand binding. Taking into account the size of the probes, a direct interaction between the probe and the ligand might be responsible of the observed effect. However, some experimental arguments indicate that this hypothesis must be discarded. First, a direct interaction should lead to a competition between the probe and the ligand. Unexpectedly, the K, values for the two ligands were significantly lower with the labeled protein than with the free protein. This increased affinity cannot be easily explained by any direct interaction between the

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M. DESMADRIL ET AL.

Fig. 5. PGK structure showing the distances between the mutated residues and the nearest atom of the ligand. Backbone of residues 1-10 and 406-415 is shown in purple.

probe and the ligands since the same effect was observed whatever the location of the probe in the cleft. Second, whereas the size of the probe is fairly large, for some SH locations (C97A,A377C)the distance between the probe and the substrate binding sites is too large to allow a direct interaction. Indeed, as shown in Figure 5, a straight line drawn from residues 376 and 183 and the binding sites corresponds, respectively, to 11.6 and 17.4 A and passes through the structure. When AEDANS is linked to Cys 35, the distance is of the same order of magnitude as the size of the probe (11.6 A). However, the dissociation constant for 3PG does not indicate the existence of interaction between the substrate and the probe. The last argument is certainly the most important. For the mutant protein C97A,A377C,the binding of each ligand separately does not lead to any change of the fluorescence properties of the probe. Only the ternary complex had a fluorescence intensity emission lowered to 50% with respect to the

ligand-free protein. It is clear that in this case, the observed changes in the fluorescence properties cannot be related to direct interaction between the probe and the ligand. Thus the changes in the environment of the probe suggest that conformational changes did occur upon ligand binding. Taking into account all the results obtained by studying the change in enzymic activity following SH labeling, it can be concluded that, in the presence of the probe, these changes are not efficient enough to bring the catalytic cycle to completion. The comparison of the data obtained for the reactivities of the thiol groups on the one hand, and the fluorescence properties of AEDANS on the other hand provides complementary information. Whereas 3PG binding did not lead to a large effect on the thiol group reactivity of mutant C97A,S35C, it significantly modified the fluorescence properties of the probe linked to this residue. Such a difference could be explained by the distance between the naphtalen ring and the thiol group itself. Whereas

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the immediate vicinity of the thiol remained accessible following 3PG binding, the fluorescent probe shifted from a hydrophilic environment to a hydrophobic one (see Fig. 2A and Table Iv), suggesting that the probe became buried. In contrast, the probe linked to the thiol group of mutant protein C97A,A183C experienced a change that drove the probe into a more hydrophilic environment upon 3PG binding. The probe grafted on Cys 183 (mutant C97A, A183C) should be in contact mainly with the C-end of the polypeptide chain, including helix XIV (406415). This stretch comes back from the C-domain and packs on the N-domain. It has been shown that a deletion of this region dramatically affects the catalytic properties. The present results suggest that the conformational changes occurring upon ligand binding imply a reorganization of the interactions between this C-terminal region and the N-terminal domain. These results must be reconciled with the hypotheses proposed to account for the conformational change induced by ligands. For yeast PGK” as well as for horse muscle PGK,lS it has been proposed that the hinge bending motion involves helix V (7 for the horse enzyme), the motion resulting from a “helix scissors” involving helices 7 and 14 for the horse enzyme. The results obtained with AEDANS seem to support this hypothesis, since a rotation of these two helices could move the fluorescent probes in opposite ways. A clear conclusion from the present work is that the observed changes mainly involve the part of the protein located in the cleft, particularly the environment surrounding residues 35 and 183, within a sphere of about 10 A, which is the size of the AEDANS probe. No alteration was observed on the external side of the protein, as reflected by the absence of variation in the properties of residues 140 and 249. Although a conformational change was already suggested by previous it was never clearly shown to what extent these changes are required for the catalytic process. Here, it is clearly shown that when there is an impediment to these conformational changes, there is a loss in the enzymatic activity, although the two ligands are bound to the protein. It is interesting to note that only 3PG induced the observed conformational changes. With protein mutant C97A,A377C, the binding of the two ligands was required to produce a modification in the fluorescence of AEDANS, whereas the reactivity of the thiol group decreased on binding each ligand separately. The difference between the data obtained from fluorescent probe and thiol reactivity is probably related to the distance separating the thiol group and the naphtalen ring. According to the present results, the ambiguity noted in the literature concerning the nature of the ligand triggering

the conformational changes, can be explained by the differences in the methods and/or probes used. It is likely that substrates separately and together induce discrete molecular motions, which may or may not be observed depending on the experimental approach.

ACKNOWLEDGMENTS This work was supported by the Centre National de la Recherche Scientifique, by a grant from the Ministere de la Recherche et de la Technologie (no. 496), by Fondation pour la Recherche MBdicale Francaise, and by a contract with the Centre National de Transfusion Sanguine. REFERENCES 1. Fifis, T., Scopes, R.K. Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography. Biochem. J. 175:311-319, 1978. 2. Scopes, R.K. Phosphoglycerate kinase In: “The Enzymes,” vol. 8,3rd ed, Boyer, P.D. (ed.). New York: Academic Press, 1973:335-351. 3. Bryant, T.N., Watson, H.C., Wendell, P.I. Structure of Yeast phosphoglycerate kinase. Nature (Lond.) 247:1417, 1974. 4. Watson, H.C., Walker, N.P.C., Shaw, P. J., Bryant, T. N., Wendell, P.L., Fothergill, L.A., Perkins, R.E., Conroy, S.C., Dobson, M.J., Tuite, M.F., Kingsman, A.J., Kingsman, S.M. Sequence and structure of yeast phosphoglycerate kinase. EMBO J., 1 :1635-1640, 1982. 5. Blake, C.C.F., Evans, P.R. Horse muscle phosphoglycerate kinase. J. Mol. Biol. 84:585-601, 1974. 6. Banks, R.D., Blake, C.C.F., Evans, P.R., Haser, R., Rice, D.W., Hardy, G.H., Merrett, M., Phillips, A.W. Sequence, structure and activity of PGK. A possible hinge bending enzyme. Nature (Lond.) 279:773-777, 1979. 7. Blake, C.C.F., Rice, D.W. Phosphoglycerate kinase. Phil. Trans. R. SOC.Lond. A. 293:93-104,1981, 8. Pickover, C. A., Mc Kay, D.B., Engelman, D.M., Steitz, T. A. Substrate binding closes the clefi between the domains of yeast phosphoglycerate kinase. J. Biol. Chem. 254:11323-1 1329,1979. 9. Sinev, M.A., Razgulyagev, 0. I., Vas, M., Timchenko, A. A., Ptitsyn, O.B. Correlation between enzyme activity and hinge bending domain displacement in 3-phosphoglycerate kinase. Eur. J . Biochem 180:61-66, 1989. 10. Wilson, H.R., Williams, R.J.P., Littlechild, J.A., Watson, H.C. NMR analysis of the interdomain region of yeast phosphoglycerate kinase. Eur. J. Biochem. 170:529-538, 1988. 11. Fairbrother W.J., Walker, P.A., Minard P., Littlechild, J.A., Watson, H. C., Williams, R.J.P. NMR analysis site specific mutant of Yeast phosphoglycerate kinase. Eur. J. Biochem. 183:57-67, 1989. 12. Wrobel, J.A., Stinson, R.A. Anion binding to yeast phosphoglycerate kinase. Eur. J. Biochem 85345-350, 1978. 13. Krietch, W.K.G., Biicher, T. 3 phosphoglycerate kinase from rabbit skeletal muscle and yeast. Eur. J. Biochem. 17:568-575, 1970. 14. Cserpan I., Vas, M. Effects of substrates on the heat stability and the reactivities of thiol groups of 3-phosphoglycerate kinase. Eur. J . Biochem. 131:157-162, 1983. 15. Wilson, C.A.B., Hardman, N., Fothergill-Gilmore, L.A., Gamblin S.J., Watson, H. C. Yeast phosphoglycerate kinase: Investigation of catalytic function by site directed mutagenesis. Biochem J . 241:609-614, 1987. 16. Mas, M.T., Resplander, Z.E., Riggs, A. D. Site directed mutagenesis of glutamate 190 in the hinge region of yeast 3-phosphoglycerate kinase: Implications for the mechanism of domain movement. Biochemistry 265369-5377, 1987. 17. Mas, M.T., Bailey, J.M. Resplander, Z.E. Site directed mutagenesis of Histidine 388 in the hinge region of yeast 3-phosphoglycerate kinase: Effects on catalytic activity

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