An NMR analysis of the binding of inhibitors to yeast phosphoglycerate kinase

The binding of a series of inhibitors to the enzyme phosphoglycerate kinase has been studied using NMR to uncover the binding sites and the effects of binding on the protein conformation. The very effective inhibitor, Suramin, causes the most pronounced changes. The design of inhibitors for mobile proteins is discussed.     

NMR analysis of the interdomain region of yeast phosphoglycerate kinase

Previous proton and phosphorus nuclear magnetic resonance studies with yeast phosphoglycerate kinase have been extended using a higher-resolution spectrometer and a greater variety of binding agents. The new study shows that, apart from a few isolated mobile side chains distributed over the protein surface, there is a mobile section of phosphoglycerate kinase associated with the inter-domain region of the molecule. This region gives relatively well resolved resonances which are quite distinct from those originating from the remainder of the protein. This suggests that the molecule fluctuates between many states including several open or substrate binding forms in addition to the closed and supposedly catalytically competent form of the enzyme. The occupancy of these states appears to be affected by several anions including sulphate, phosphate and cobalticyanide, as well as substrates and their analogues.     

An NMR study of anion binding to yeast phosphoglycerate kinase

Anion binding to yeast phosphoglycerate kinase has been investigated using 1H-NMR spectroscopy. The use of anionic paramagnetic probes. [Cr(CN)6]3- and [Fe(CN)6]3-, has enabled the location of the primary anion binding site in the 'basic-patch' region of the amino-terminal domain. The anions interact most closely with Arg-65 and Arg-168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3-phosphoglycerate. Binding of 3-phosphoglycerate and 1.3-bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity. Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations while the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.     

One- and two-dimensional NMR studies of yeast phosphoglycerate kinase

One- and two-dimensional proton NMR studies have been carried out on yeast phosphoglycerate kinase (Mr approximately 45,000) in order to identify amino-acid spin systems and obtain sequence-specific assignments. A number of sequence-specific assignments have been made using a combination of structural information contained in nuclear Overhauser effect spectra and X-ray crystallographic data. The results of substrate binding studies (both 3-phosphoglycerate and Mg.ATP), which indicate mutual reorientation of certain assigned aromatic residues in the inter-domain region of the protein, are discussed.     

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.     

An engineered amino-terminal domain of yeast phosphoglycerate kinase with native-like structure.

Previous studies have suggested that the carboxy-terminal peptide (residues 401-415) and interdomain helix (residues 185-199) of yeast phosphoglycerate kinase, a two-domain enzyme, play a role in the folding and stability of the amino-terminal domain (residues 1-184). A deletion mutant has been created in which the carboxy-terminal peptide is attached to the amino-terminal domain (residues 1-184) plus interdomain helix (residues 185-199) through a flexible peptide linker, thus eliminating the carboxy-terminal domain entirely. CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N-domain is soluble, monomeric, compactly folded, native-like in structure, and capable of binding the substrate 3-phosphoglycerate with high affinity in a saturable manner. The midpoint of the guanidine-induced unfolding transition was the same as that of the native two-domain protein (Cm approximately 0.8 M). The free energy change associated with guanidine-induced unfolding was one-third that of the native enzyme, in agreement with previous studies that evaluated the intrinsic stability of the N-domain and the contribution of domain-domain interactions to the stability of PGK. These observations suggest that the C-terminal peptide and interdomain helix are sufficient for maintaining a native-like fold of the N-domain in the absence of the C-domain.     

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.     

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.     

Photochemically induced dynamic nuclear polarization NMR study of yeast and horse muscle phosphoglycerate kinase

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified 1H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP. Identification of the CIDNP-enhanced resonances with respect to the type of amino acid residue has been achieved since only tyrosine yields emission peaks and the dye 8-aminoriboflavin enhances tryptophan but not histidine. By use of the known amino acid sequences and structures derived from X-ray crystallographic studies of the enzymes from the two species, assignment of the specific residues in the protein sequences giving rise to the CIDNP spectra was partially achieved. In addition, flavin dye accessibility was used to probe any changes in enzyme structure induced by substrate binding. The nine resonance peaks observed in the CIDNP spectrum of yeast phosphoglycerate kinase have been assigned tentatively to five residues: histidines-53 and -151, tryptophan-310, and tyrosines-48 and -195. The accessibility of a tyrosine to photoexcited flavin is reduced in the presence of MgATP. Since the tyrosine residues are located some distance from the MgATP binding site of the catalytic center, it is proposed either that this change is due to a distant conformational change or that a second metal-ATP site inferred from other studies lies close to one of the tyrosines. Horse muscle phosphoglycerate kinase exhibits seven resonances by CIDNP NMR.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site.

Exchange-inert beta, gamma-bidentate Cr(H2O)x(NH3)y ATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min-1 to 0.118 min-1 for Cr(NH3)4ATP and Cr(H2O)4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k(obs) by 3-PGA was proportional to the number of NH3 ligands present in the coordination sphere of Cr3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H2O)4ATP. delta diastereomer of Cr(H2O)4ATP yielded an order of magnitude smaller Ki value compared to the value observed with the lambda isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H2O)4ATP or Cr(NH3)4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme CrATP and enzyme.CrATP.3-PGA complexes.     

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.     

Substrate binding closes the cleft between the domains of yeast phosphoglycerate kinase.

Using small angle x-ray scattering from solutions of yeast phosphoglycerate kinase, we have measured the radius of gyration of the enzyme both in the presence and in the abscence of ligands. We find that the radius of gyration decreases by 1.09 +/- 0.34 A upon binding both substrates MgATP and 3-phosphoglycerate to form the ternary complex. Smaller decreases, at the limit of the precision of the measurement, were found for the separate binding of MgATP (0.30 +/- 0.50 A). Using computer modeling, it has been estimated that a substrate-induced cleft closure in phosphoglycerate kinase resulting from one lobe rotating 8-14 degrees relative to the other lobe lobe is consistent with this observed change in radius of gyration. We suggest, therefore, that the conformational change that results in the smaller radius of gyration for the ternary complex is a hinge motion of the two lobes which produces a closing of the cleft between the two lobes. The apparent similarity of the ligand-induced change in phosphoglycerate kinase to the cleft closure in hexokinase suggests that this kind of conformational change may prove to be a rather general kinase phenomenon (Bennett, W.S., and Steitz T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852; Anderson, C.M., Zucker, F.H., and Steitz, T.A. (1979) Science 204, 375-380).