Site-directed mutagenesis of yeast phosphoglycerate kinase. The 'basic-patch' residue arginine 168

There is evidence, some of it of questionable authenticity, which suggests that phosphoglycerate kinase takes up a more compact form following the binding of substrates. Using this evidence it has been assumed that a conformational rearrangement is required for phosphoryl transfer to occur and that this is brought about by moving the enzyme's two domains towards each other. In order to test this hypothesis we have modified, by site-directed mutagenesis, an arginine residue thought to be involved in stabilising the transition-state intermediate. Although some 1.3 nm away from the site of phosphoryl transfer, as seen in the crystallographically determined structure, the substitution of arginine 168 by lysine (R168K) more than halves the specific activity of the enzyme. Substituting the arginine with a methionine (R168M) reduces activity further, but not completely, thus proving that the charge associated with this residue is not essential for catalytic activity. Both mutations raise the Michaelis constants (Km) for ATP and glycerate 3-phosphate. The largest change is observed with the triose substrate and the methionine mutant, suggesting that the primary function of arginine 168 is to influence the environment of this substrate. The effect on activity of adding sulphate to R168K and R168M mutant enzyme has also been investigated. The sulphate activation effect at low substrate concentrations is reduced for the methionine substitution but almost abolished for the lysine substitution. The most reasonable explanation of all these findings is that, in the wild-type enzyme, the guanidinium group of arginine 168 forms a hydrogen bond with one of the triose substrate's C1 oxygens. This steric arrangement would not be possible in the 'open form' of this enzyme as observed in the crystal structure.     

Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase.

Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme.     

Site-directed mutagenesis of yeast phosphoglycerate kinase Arginines 65, 121 and 168

In the absence of a structure of the closed form of phosphoglycerate kinase we have modified by site directed mutagenesis several of the residues which, on the basis of the open form structure, are likely to be involved in substrate binding and catalysis. Here we report on the kinetic and anion activation properties of the yeast enzyme modified at positions 65, 121 and 168. In each case an arginine, thought to be involved in the binding of the sugar substrate's non-transferable phosphate group, has been replaced by lysine (same charge) and by methionine (no charge). K_m values for 3-phosphoglycerate of all six mutant enzymes are only marginally higher than that of the wild-type enzyme. Removing the charge associated with two of the three arginine residues appears to influence (as judged by the measured K_m's) the binding of ATP. Although binding affinity is not necessarily coupled to turnover the substitutions which have the greatest effect on the K_m's do correlate with the reduction in enzymes maximum velocity. The one exception to this generalisation is the R65K mutant which, surprisingly, has a significantly higher k_cat than the wild-type enzyme. In the open form structure of the pig muscle enzyme each of the three substituted arginines residues are seen to make two hydrogen bonds to the sugar substrate's non-transferable phosphate. From this it might be expected that anion activation would be similarly affected by the substitution of any one of these three residues. Although the interpretation of such effects are complicated by the fact that one of the mutants (R65M) unfolds at low salt concentrations, this appears not to be the case. Replacing Arg¹²¹ and Arg¹²¹ with methionine reduces the anion activation whereas a lysine in either of these two positions practically destroys the effect. With the substitutions at residue 65 the opposite is observed in that the lysine mutant shows anion activation whereas the methionine mutant does not.     

Anion binding to yeast phosphoglycerate kinase.

The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.     

Efficient expression and characterization of isolated structural domains of yeast phosphoglycerate kinase generated by site-directed mutagenesis

The two domains of yeast phosphoglycerate kinase were produced by recombinant techniques. The N-domain was obtained by the introduction of a termination codon at the position coding for Phe185, and the C-domain by a deletion in the gene of the coding sequence between Ser1 and Leu186. Both domains were efficiently expressed in yeast, the level for the C-domain being greater than that for the N-domain. Both domains were found to have a quasi-native structure; the C-domain retained its ability to bind nucleotides. Small local differences were detected in domain structure compared to that in the whole enzyme, probably due to the lack of interdomain stabilizing interactions. Nevertheless, such an approach provides direct evidence for independent folding of domains in a two-domain protein.     

Sequence and structure of yeast phosphoglycerate kinase.

The structure of yeast phosphoglycerate kinase has been determined with data obtained from amino acid sequence, nucleotide sequence, and X-ray crystallographic studies. The substrate binding sites, as deduced from electron density maps, are compatible with known substrate specificity and the stereochemical requirements for the enzymic reaction. A carboxyl-imidazole interaction appears to be involved in controlling the transition between the open and closed forms of the enzyme.     

Yeast phosphoglycerate kinase: investigation of catalytic function by site-directed mutagenesis.

A salt link buried in the domain interface of phosphoglycerate kinase has been implicated as being important in controlling the conformational transition from the open, or substrate-binding, to the closed, or catalytically competent, form of the enzyme. The residues contributing to the salt link are remote from the active site, but are connected to the substrate-binding sites through strands of beta-sheet. It has been suggested that these residues may also mediate sulphate and anion activation. These assumptions have been tested by examining the properties of a site-directed mutant (histidine-388----glutamine-388). The expression and overall structural integrity of the mutant, produced in yeast from a multicopy plasmid, remains essentially unaltered from the wild-type enzyme. However, the mutant enzyme has a kcat. reduced by 5-fold. The Km for ATP is lowered by 3-fold, and the Km for 3-phosphoglycerate is unaffected. The effects of sulphate on activity over a wide range of substrate concentrations appear to be the same for both the mutant and wild-type enzymes. These results lead to a reappraisal of the mechanistic role of the inter-domain histidine-glutamate interaction, as well as a refinement of the kinetic model of the enzyme.     

Thermodynamic study of yeast phosphoglycerate kinase

Enthalpies of binding of MgADP, MgATP, and 3-phosphoglycerate to yeast phosphoglycerate kinase have been determined by flow calorimetry at 9.95-32.00 degrees C. Combination of these data with published dissociation constants [Scopes, R.K. (1978) Eur. J. Biochem. 91, 119-129] yielded the following thermodynamic parameters for the binding of 3-phosphoglycerate at 25 degrees C: delta Go = -6.76 +/- 0.11 kcal mol-1, delta H = 3.74 +/- 0.08 kcal mol-1, delta So = 35.2 +/- 0.6 cal K-1 mol-1, and delta Cp = 0.12 +/- 0.32 kcal K-1 mol-1. The thermal unfolding of phosphoglycerate kinase in the absence and presence of the ligands listed above was studied by differential scanning calorimetry. The temperature of half-completion, t 1/2, of the denaturation and the denaturational enthalpy are increased by the binding of the ligands, the increase in t 1/2 being a manifestation of Le Chatelier's principle and that in enthalpy reflecting the enthalpy of dissociation of the ligand. Only one denaturational peak was observed under all conditions, and in contrast with the case of yeast hexokinase [Takahashi, K., Casey, J.L., & Sturtevant, J.M. (1981) Biochemistry 20, 4693-4697], no definitive evidence for the unfolding of more than one domain was obtained.     

Enzymatic activity of immobilized yeast phosphoglycerate kinase

This work reports the first evidence that recombinant yeast phosphoglycerate kinase (PGK) is still significantly active when immobilized on glass and muscovite mica. Using previous work to improve the sensitivity of the existing setup, Tapping Mode atomic force microscopy (AFM) was used in a liquid environment to determine the surface enzyme coverage of derivatized mica and glass slides. When associated to spectrophotometric measurements, the AFM data allows assessing the catalytic constant of surface enzymes and comparing it to bulk values. The validity of the Michaelis-Menten model for surface reactions is discussed, supported by spectroscopic measurements of the surface consumption of 1,3-bis-phosphoglycerate (1,3-BPG). Only a few percent of the enzyme material maintains its initial bulk activity. This value could constitute a guideline for biosensors made with the method used here whenever a rapid assessment of the remaining surface activity is needed.     

The complete amino acid sequence of yeast phosphoglycerate kinase.

The complete amino acid sequence of yeast phosphoglycerate kinase, comprising 415 residues, was determined. The sequence of residues 1-173 was deduced mainly from nucleotide sequence analysis of a series of overlapping fragments derived from the relevant portion of a 2.95-kilobase endonuclease-HindIII-digest fragment containing the yeast phosphoglycerate kinase gene. The sequence of residues 174-415 was deduced mainly from amino acid sequence analysis of three CNBr-cleavage fragments, and from peptides derived from these fragments after digestion by a number of proteolytic enzymes. Cleavage at the two tryptophan residues with o-iodosobenzoic acid was also used to isolate fragments suitable for amino acid sequence analysis. Determination of the complete sequence now allows a detailed interpretation of the existing high-resolution X-ray-crystallographic structure. The sequence -Ile-Ile-Gly-Gly-Gly- occurs twice in distant parts of the linear sequence (residues 232-236 and 367-371). Both these regions contribute to the nucleoside phosphate-binding site. A comparison of the sequence of yeast phosphoglycerate kinase reported here with the sequences of phosphoglycerate kinase from horse muscle and human erythrocytes shows that the yeast enzyme is 64% identical with the mammalian enzymes. The yeast has strikingly fewer methionine, cysteine and tryptophan residues.     

Conversion of yeast phosphoglycerate kinase into amyloid-like structure

Yeast phosphoglycerate kinase is a structurally well-characterized enzyme consisting of 415 amino acids without disulfide bonds. Anion-induced refolding from its acid-unfolded state gives rise to the formation of worm-like amyloid fibrils with a persistence length of 73 nm. Electron microscopy and small-angle X-ray scattering data indicate that the fibrils have an elliptical cross-section with dimensions of 10.2 nm x 5.1 nm. About half of all amino acids are organized in form of cross-beta structure which gives rise to typical infrared spectra, X-ray diffraction and yellow-green birefringence after Congo red staining. The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X-ray scattering, was found to be strongly dependent on protein concentration. The infrared data indicate that the formation of cross-beta structure practically comes to an end already after some hours, whereas the length-growth of the amyloid fibrils, monitored by small-angle X-ray scattering, was not yet completed after 1,300 hours.     

Nuclear-magnetic-resonance study of the active-site structure of yeast phosphoglycerate kinase.

The enzyme 3-phosphoglycerate kinase from yeast has been studied by observation of the proton nuclear magnetic resonance spectrum at 270 MHz using Fourier transform techniques. Difference spectroscopy was used to enhance the resolution and to identify specific ligand binding effects and conformational changes. Perturbations involving single protons of amino-acid residues could thus be detected despite the relatively high molecular weight of the protein (47000), particularly in the aromatic (6-9 ppm) and methylene (2-3 ppm) regions of the spectrum.     

Binding of substrates and other anions to yeast phosphoglycerate kinase.

1. The binding of all four substrates to yeast phosphoglycerate kinase has been studied using a gel filtration technique. The binding of phosphate and sulphate anions has also been investigated. 2. Two sites for each adenine nucleotide were found, one site being weaker than the other by between 30 and 50-fold. Only one binding site for the phosphoglycerate substrates was found. 3. 1,3-Bisphosphoglycerate (1,3-P2-glycerate) bound to the enzyme approximately 1000 times tighter than the other three substrates, its dissociation constant being 0.06 micrometer at ionic strength 0.15 M. 4. Sulphate and phosphate were mutually competitive and sulphate competed with the binding of all substrates except MgADP. MgADP bound to the enzyme more weakly in the presence of sulphate. The dissociation constant for sulphate binding was 1.6 mM at ionic strength of 0.15 M, and 0.05 mM at ionic strength 0.015 M. 5. These results are consistent with sulphate acting as a competitive inhibitor, as found by kinetic studies at high sulphate concentrations. The activatory effect of sulphate at lower concentrations and the substrate activation phenomea displayed by this enzyme, are interpreted in terms of a two-step dissociation of 1, 3-P2-glycerate. The presence of moderate concentrations of MgATP, 3-phosphoglycerate or sulphate causes acceleration of the rate of dissociation of the product, 1, 3-P2-glycerate, this being the rate-limiting step in the overall enzyme reaction.     

Pressure-temperature-induced denaturation of yeast phosphoglycerate kinase, a double-domain protein.

Pressure-induced denaturation of yeast phosphoglycerate kinase was studied at various temperatures, as a model double-domain protein, using intrinsic fluorescence, 4th derivative absorbance, CD, and DSC. A thermodynamic transition intermediate was observed in the pressure-denaturation, as was reported for the cold denaturation. From the different response of Trp and Tyr residues, as monitored by fluorescence and 4th derivative absorbance changes, the C-terminal domain carrying all the Trp residues seemed to exert structural changes at relatively lower pressure. A further structural change involving both domains was observed at higher pressures. The two-step changes occurred almost simultaneously during heat denaturation.     

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK, has been constructed which greatly facilitates the insertion and removal of mutant enzyme genes by cleavage at newly introduced BamHI sites. This vector has been used to prepare mutant protein in appreciable (100 mg) quantities for use in kinetic, crystallographic and NMR experiments. Aspartate 372 is an invariant amino acid residue in genes known to code for a functionally active PGK. The function of this acidic residue appears to be to help desolvate the magnesium ion complexed with either ADP or ATP when this substrate binds to the enzyme. Both crystallographic and nuclear magnetic resonance experiments show that the replacement of the residue with asparagine has only minimal effects on the overall structure. The substitution of the charged carboxyl group with that of the neutral amide affects the binding of the nucleotide substrate as predicted but not, as might have been expected, the binding of 3-phosphoglycerate. The overall velocity of the enzymic reaction (Vmax) is reduced 10-fold by the substitution of aspartic acid 372 by an asparagine residue (D372N). This reduction in Vmax is considerably less than one would expect from its known position within the structure of the enzyme. This result therefore poses questions about our understanding of charged groups at the active centres of enzymes and of the reason for their apparent conservation.