Article

Electrophoretic Purification as well as Some Physical and Chemical Characterizations of Phosphoglycerate Kinase from Yeast

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Abstract

1The commercially available crystalline phosphoglycerate kinase from yeast was found to be a mixture of enzymically active and inactive proteins. These different protein components can be separated by column electrophoresis. Besides a large fraction (about 50% of the material) without phosphoglycerate kinase activity three enzymically active electrophoretic components appeared.2Sedimentation and sedimentation equilibrium studies indicated that the proteins of the main active component and one of the two smaller active components are homogeneous and of the same size. The molecular weight was estimated to 44600 ± 1600.3The amino acid analyses indicated that the amino acid compositions of these two protein components are the same. From these analyses the nitrogen and sulphur contents were estimated to about 16.96% and 0.29%, respectively. E1%1cm at 280 nm is close to 0.50. The amino acid composition shows also that if the enzyme consists of subunits, these cannot all be identical.4The specific activity of the two similar protein components was estimated to 990 units/mg, when measured in the direction of 1,3-diphosphoglycerate formation.

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... Crystalline phosphoglycerate kinase from yeast was obtained from Boehringer Mannheim GmbH (Mannheim, Germany). The enzyme was further purified by column electrophoresis [S] and the main electrophoretic component I I B [8] used. ...
... The volume of the solution on each side of the dialysis membrane was I .5 ml. A molecular weight of 45000 [8] and a n extinction coefficient of 0.5 mg-l x ml x cm-l [I, 81 were used t o estimate the molar concentrations of phosphoglycerate kinase. ...
Article
The present study shows that MgATP2- or MnATP2- as substrates of phosphoglycerate kinase can be replaced by CaATP2-, ZnATP2-, CoATP2- or NiATP2-. MnATP2- and NiATP2- are about 90% and 15%, respectively, and the other ATP4--metal ion complexes roughly 70% as good substrates as MgATP2-. No measurable activity was found with Be(II) or Fe(III). The effectiveness of the different substrate species appears to be determined by factors such as the size and polarizing capability of the metal ion as well as of the structure of the relevant metal ion complex and the rate of ligand dissociation processes. Detailed kinetics with Zn(II), Mn(II) and Co(II) showed that: (a) Zn2+ is a strong uncompetitive inhibitor of ZnATP2-, Ki approx. 0.02 mM. (b) Mn2+ is a competitive inhibitor of MnATP2- at concentrations < 0.1 mM, Ki approx. 2.3 mM. This inhibition is dependent on the 3-P-glycerate concentration. At about 1 mM and higher concentrations Mn2+ acts as an uncompetitive inhibitor of MnATP2-. (c) Co2+ is a competitive inhibitor of CoATP2- at about 1 mM and higher concentrations, Ki approx. 3 mM. With CoATP2- as substrate the activity is slightly increased in the presence of free Co2+ and/or free ATP4- at concentrations < 0.5 mM. When the CoATP2- concentration is varied, the activity seems not to become constant at concentrations ten times the apparent Michaelis constant for CoATP2-. Equilibrium dialysis studies on the binding of Co2+ to the enzyme showed that this ion binds more strongly to the enzyme than, for example, Mn2+. This probably explains the differences in activation and inhibition observed with these two ions. The kinetic patterns obtained with the different metal ions indicate that Ni(II) behaves as Co(II), Cd(II) as Zn(II) and Ca(II) as Mg(II), which is somewhat similar in behaviour to Mn(II).
... That all mammalian phosphoglycerate kinases so far analysed contain 12 [5,31] or 13 [6,2,32,33] methionine residues for a molecular weight of 47 000 is remarkable, especially considering that there is only one triplet codon for methionine in the genetic code. Overall the mammalian phosphoglycerate kinase amino acid analyses are very similar and except for sulphurcontaining amino acids, so is yeast [2,34]. The similarity of structure of the yeast and horse muscle enzymes from X-ray studies [35,36] further confirm that the basic structure and composition of phosphoglycerate kinase has been strongly conserved throughout evolution. ...
Article
1. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from 'normal' phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described. 2. The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources. 3. Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3-phosphoglycerate and MgADP), energy of activation and thermal denaturation. 4. The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1--2 more positive charges than the A. 5. Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis. 6. The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.
... The crystallized enzyme ran as a single component on starch-gel electrophoresis, though as with other phosphoglycerate kinases, with considerable streaking towards the anode (Scopes, 1968). There was no evidence of the minor components found by Larsson-Rainikiewicz (1970). The enzyme also gave only a single band on polyacrylamide-sodium dodecyl sulphate-gel electrophoresis, at a position corresponding to 50000daltons. ...
Article
3-Phosphoglycerate kinase has been isolated from yeast by a new procedure. Over 1g was obtained from 450g of granulated baker's yeast; it had a specific activity of up to 940units/mg at 30 degrees C. Six distinct crystalline forms have been grown, at least one of these being suitable for X-ray diffraction studies. The crystalline preparation is pure, judged by starch-gel or sodium dodecyl sulphate-polyacrylamide-gel electrophoresis; the latter method indicating that the enzyme is monomeric, with a molecular weight near to 50000.
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Yeast phosphoglycerate kinase is irreversibly inactivated upon incubation with 5'-[p-(fluorosulfonyl)-benzoyl]-1-N6-ethenoadenosine (5'-FSB epsilon A), an analogue to the nucleotide substrate. Marked protection against inactivation occurs with MgATP, ATP, MgADP, ADP, and 3-phosphoglycerate, suggesting that a part of the catalytic center is modified. The time dependence of the inactivation is characterized by a nonlinear kinetic profile. Curve fitting of various models for ligand binding to the enzyme suggested a two-site model. Modification of one of the sites appears to protect the catalytically essential site from modification. Stoichiometric studies show that the relationship between moles of 5'-FSB epsilon A incorporated per mole of enzyme and the residual enzymatic activity also shows nonlinear behavior. An extrapolated value of 1.5 mol of bound label/mol of enzyme corresponds to complete inactivation. The apparent overall pseudo first-order rate constant for the reaction between phosphoglycerate kinase and 5'-FSB epsilon A, as well as the separate rate constants for the modification, exhibit saturation behavior with respect to the concentration of 5'-FSB epsilon A, indicative of a rapid reversible binding of the reagent to the enzyme prior to modification.
Article
The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-phosphoglycerate kinase (EC 2.7.2.3), present in an extract of Bakers' yeast, are largely kept in solution by minor amounts of polyethylene glycol-bound triazine dyes (Procion yellow HE-3G and Procion olive MX-3G) even when the solution contains such concentrations of polyethylene glycol (12.5% w/w) which normally precipitate the enzymes. The specific prevention from precipitation can be used for purification of enzyme, preferentially in dealing with crude extracts, which has been demonstrated in this work. A 3.4-fold purification of glucose-6-phosphate dehydrogenase has been achieved with good recovery (93%). Further purification has been possible by combining the recovered (enzyme-containing) supernatant liquid with a solution of dextran which generates an aqueous two-phase system. The lower, dextran-containing phase extracts part of the remaining bulk proteins leaving the target enzyme in the upper phase. The advantages of this method for enzyme purification in large scale are discussed.
Article
1.1. Purified muscle proteins have been run on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular sizes of their subunits were determined by comparison with marker proteins.2.2. Myofibrils were dissolved in sodium dodecyl sulphate, and separated into the constituent subunits on electrophoresis. The main components of the spectrum obtained were identified, and their sizes determined. In addition there was indication of the presence of a protein of subunit size 105 000 molecular weight which has not previously been described.3.3. Sarcoplasmic glycolytic enzymes were also studied. Only two of these (phosphofructokinase and phosphoglucose isomerase) gave subunit sizes in this system significantly different from accepted values, in both cases the new values are nearly 20% lower. It is not clear whether these proteins behave anomalously in dodecyl sulphate electrophoresis, or whether the more conventional techniques for determining molecular sizes are in error.
Article
A phosphoryl-enzyme intermediate as part of the mechanism of phosphoglycerate kinase has been suggested for the rabbit muscle enzyme (6) and the yeast enzyme (7,8). ATP in the binary enzyme-substrate complexes appeared to phosphorylate these enzymes and ADP-ATP exchange activities were observed (6,7,8). The present report shows, however, that highly purified yeast enzyme cannot be phosphorylated by ATP. On the other hand ADP-ATP exchange activity was obtained but this was proportional to trace amounts of adenylate kinase activity, which was found to contaminate the enzyme preparations. Thus a Ping Pong mechanism as an alternative to a mechanism including a ternary complex between the enzyme and its two substrates appears very improbable. Whether the enzyme or the phosphoryl-group-accepting substrate is responsible for the primary nucleophilic attack occurring in the ternary complex is still an open question, however. Yeast phosphoglycerate kinase appears to have no ATPase activity.
Article
A 3.5 Å resolution electron density map of yeast phosphoglycerate kinase has been calculated from which a skeletal model of the enzyme's single polypeptide chain has been constructed. The position of the nucleotide substrate ADP when bound to the enzyme has been defined.
Article
Equilibrium studies on ATP4− and 3-P-glycerate binding to phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed. The results show that the enzyme contains two binding sites for both ligands, as was earlier suggested to explain some of the kinetic results. As long as structural information is lacking analysis of binding data can be analyzed if assumptions are made whether the sites are equivalent or nonequivalent, and whether ligation of these sites is mutually dependent or not. To understand the experimental results explicit expressions were derived for the limiting slopes and intercepts of Klotz and Scatchard plots, respectively, in terms of the intrinsic affinity constants valid under the separate assumptions made. The algebraic expressions for the limiting slopes were analyzed in order to ascertain the relationship between the form of these graphical plots, the prevailing types of binding site, and the intrinsic affinity constants. The results show that in case the equivalence of the binding sites is questionable it is necessary to be careful when the type of interaction is being determined. In certain cases it might be difficult to distinguish between positive and negative interaction. Sites that appear to be equivalent and independent could equally well be nonequivalent and dependent.
1.1. Rat skeletal muscle phosphoglycerate kinase (E.C. 2.7.2.3) has been purified to homogeneity by a six-step process.2.2. The enzyme is a single-stranded protein with a molecular mass of about 43,000 Daltons as determined by exclusion chromatography.3.3. The monomeric nature of rat muscle phosphoglycerate kinase (PGK) has been substantiated by analogy with PGK's from other sources and by fingerprint analysis after tryptic digestion.4.4. Complete amino acid analysis shows exceedingly close homology with the rabbit muscle enzyme and surprising agreement with the yeast enzyme. Thus, the molecular weight and compositional data suggest that phosphoglycerate kinases from both primitive and highly differentiated cells are approximately the same size.5.5. Rat muscle PGK has an extinction coefficient, E1·0cm0·1%, at 280 nm of 0·811.6.6. Rat muscle PGK contains five free sulfhydryl groups compared to seven and one for the rabbit muscle and yeast enzymes, respectively.7.7. None of the above enzymes manifest disulfide bridges.
Article
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Human phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) was purified from red blood cells. The stepwise procedures are elimination of hemoglobin from the hemolysate by treatment with ethanol-chloroform, precipitation of the enzyme by ethanol, treatment with carboxymethyl-Sephadex, gel filtration on Sephadex G-75, treatment with diethylaminoethyl-Sephadex, and column chromatography with carboxymethyl-Sephadex. A crystalline preparation was obtained with an over-all yield of about 60%. The sedimentation patterns on analytical ultracentrifugation and the interference pattern of sedimentation equilibrium indicated a homogeneous preparation. The sedimentation constant (s20,w) was 3.35 S at a protein concentration of 0.6%. The molecular weight was estimated as 49,600 by the sedimentation equilibrium method. The enzyme could not be dissociated into smaller subunits by treatment with 5 m guanidine hydrochloride containing 2-mercaptoethanol, with maleic anhydride, or with sodium dodecylsulfate. The enzyme has N-acetylserine as NH2terminal and isoleucine as COOH-terminal. The specific activity of the crystalline preparation was 650 to 700 units per mg at pH 7.5 and at 25°. The enzyme required Mg²⁺ or Mn²⁺ for activity.
Article
The single cysteine residue present in phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) was modified with , , iodoacetate, iodoacetamide, and 5,5′-dithiobis-(2-nitrobenzoic acid). The SH group is fairly unreactive in its native state with most of these reagents. After 10 h about 60, 45, 25 and 15% of the group has reacted with N-ethylmaleimide, iodoacetamide, iodoacetate and 5,5′-dithiobis(2-nitrobenzoic acid), respectively.The modified enzyme is fully active, and the stability of the enzyme appears unaffected. ATP4, MgATP2− and , when used separately, did not protect the SH group from reaction. The kinetic parameters of MgATP2−, MgADP− and , respectively, are identical for the modified and unmodified enzymes. Consequently, the SH group does not seem to be directly located in the active site region, and the modification does not appear to influence the substrate binding.Kinetic studies with the enzyme show that Zn2+ is an uncompetitive inhibitor of ZnATP2−. A result demonstrating that the SH group is not a Zn2+ ligand as could earlier be assumed. The inhibitor constant is higher for the modified enzyme compared with the native enzyme, indicating that the modification reaction affects binding of free Zn2+ to its inhibitor site.
Article
The present study confirms the previous reports that detergents can facilitate the reactivation of guanidinium chloride (GdmCl) denatured rhodanese (Tandon, S. and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618; Tandon, S. and Horowitz, P. (1987) J. Biol. Chem. 262, 4486-4491). Here, we report the effect of the detergent, lauryl maltoside, on the reactivation of several enzymes other than rhodanese. For this study we used five different enzymes each having a single polypeptide chain, namely: adenosine deaminase; 3-phosphoglyceric phosphokinase; myokinase; 3 alpha-hydroxysteroid dehydrogenase; and phosphoglucomutase. The regain of enzyme activity was used to monitor refolding. Like rhodanese, these enzymes were denatured in 6 M GdmCl and diluted into a buffer containing various concentrations of lauryl maltoside. The effect of lauryl maltoside on reactivating these proteins depended on the specific enzyme used. For example, in the presence of lauryl maltoside, reactivation of adenosine deaminase increased to 98%, while phosphoglucomutase could not be reactivated significantly. The critical micelle concentration (CMC) of lauryl maltoside was measured under the present experimental conditions using 2-(p-toluidinyl)naphthalene 6-sulfonate (TNS) as an apolar fluorescent probe, and gave a value of 0.085 mg.ml-1 in 10 mM sodium phosphate (pH 7.4). The reactivating effect of lauryl maltoside was not generally related to its CMC. In some cases an induction period was observed before the enzyme attained its steady-state velocity. This might suggest the presence of intermediate(s) in the refolding pathway that could have been stabilized by the detergent. These findings indicate that 'non-denaturing' detergents may be useful for assisting reactivation of enzymes, although the optimum conditions will have to be determined for each individual case.
Article
This paper gives a presentation of ADP and AMP inhibition of phosphoglycerate kinase with MgATP2− and 3-phospho-d-glycerate as substrates at high and low Mg2+ concentrations and pH 7.8. The enzyme seems to contain at least two nucleotide binding sites, one presumably binding to MgATP2− and the other to ADP3−. The ADP3− binding site might bind MgADP1− also. AMP2− competes for the same form of the enzyme, probably the same site, as MgATP2−. ADP3− and MgADP1− are competive inhibitors and AMP2− is a non-competitive inhibitor of 3-P-glycerate. Values of the inhibition constant, Ki, for ADP3− at low Mg2+ level and MgADP1− at high Mg2+ level are 0.2 and 0.02 mM, respectively. The latter value is about ten times less than the expected Michaelis constant for corresponding substrate in the reverse reaction. Ki for AMP is about 2.0 mM at both low and high Mg2+ concentrations but the inhibition is stronger at a high than at a low Mg2+ level, probably caused by conformational and/or other differences of the enzyme at these two metal ion concentrations. The main catalytic reaction suits a pattern that is consistent with a rapid equilibrium random mechanism.
Article
Phosphoglycerate kinase, isolated from rabbit muscle in highly purified crystalline form, was compared in terms of chemical, physical and kinetic properties to yeast phosphoglycerate kinase. A summary of the data is presented. Both proteins are composed of a single polypeptide chain, and in electrophoresis only one band was found. The N-terminus is masked. The C-terminal amino acid of the muscle enzyme is valine. Muscle and yeast enzyme have nearly identical molecular weights, Michaelis-Menten constants, optimal pH values and Vmax. On the other hand, they differ significantly in their amino acid composition, especially in the content of sulfur and aromatic amino acids, in electrophoretic mobility and in heat stability. The muscle enzyme has an essential SH group. The single SH group of the yeast enzyme is apparently without importance for enzyme activity. Differences in the same direction as those observed with yeast and muscle phosphoglycerate kinase are discussed in connection with other glycolytic enzymes from the same organisms.
Article
Phosphoglycerate kinase catalyses the high-energy phosphoryl transfer of the acyl phosphate of 1,3-bisphosphoglycerate to ADP to produce ATP, a reaction requiring magnesium ions. The enzyme is widely distributed and apparently highly conserved as a monomer of molecular mass 45 000. X-ray studies of the enzymes from horse muscle and yeast, carried out in Oxford and Bristol respectively, have shown that the molecular structures of the two enzymes are almost identical. The most striking aspect of the structure is that the single polypeptide chain is organized into two separated domains composed of the N-terminal and C-terminal halves of the chain. Substrate binding studies and the determination of the complete amino acid sequence of the horse enzyme suggest that the nucleotide substrates and the phosphoglycerate substrates are bound to the C-domain and N-domain, respectively, in sites that are separated by about 12 A. In order to bring the two substrates together for catalysis, a hinge-bending conformational change involving helix rotation has been proposed, for which there is independent evidence from solution studies. Crystals of the ternary complex of the horse enzyme have been prepared that may contain the folded form of the enzyme.
Article
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The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding.
Article
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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.
Article
Anions and particularly sulfate are known to interact with 3-phosphoglycerate kinase and to induce an increase of its catalytic efficiency. The present work affords information on the location of the anionic site and on the conformational change produced by the sulfate binding. We have established that sulfate is able, first, to modify the environment of some critical amino acids (cysteine and arginines) located in the N-terminal half of the protein, second, to induce perturbation of aromatic residues as judged by spectrophotometry, and, third, to slightly decrease the magnitude of the Cotton effect at 233 nm. All these modifications are produced by sulfate concentrations required for the activation of the enzyme. The most striking result consists in a large change in the hydrodynamic properties of the protein upon sulfate interaction as determined by analytical ultracentrifugation studies. Thus, sulfate modifies the shape of the molecular, causing it to become more compact. Furthermore, a study of the binary and ternary complexes between yeast 3-phosphoglycerate kinase and its substrates suggests that such a change of the shape of the molecular only occurs in sulfate-enzyme with or without substrates and in ATP (with or without Mg2+)-3-phosphoglycerate-enzyme complexes.
Article
A kinetic study of the effects of SO4(2-) in the activity of phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) is presented. SO4(2-) behaves both as an activator and inhibitor of the reaction. Activation does not appear to affect binding of one or the other of the two substrates to the catalytic centre. As an inhibitor SO4(2-) competes with both the substrates. Thus, each substrate can constrict SO4(2-) from the inhibitor binding site, probably the catalytic centre. Under these conditions activation becomes more and more evident. There appear to exist at least two SO4(2-) binding sites, one which earlier has been defined as an anion binding site, and a second, being the catalytic centre. The former seems to have a higher affinity for SO4(2-) than the latter.
Article
In this article we look in depth at the properties of a kinase, phosphoglycerate kinase (PGK). The object of the account is to seek common features of controlled phosphate transfer reactions not only in kinases, but also in ATP-utilising pumps and in ATP synthases found in bio-energy capture devices (Table 1). We started work in this area using PGK as a model for the F, section of ATP synthase [1]. The value of PGK today in this model study rests on the following features. (a) It is of sufficient size to demonstrate both phosphate transfer and the control mechanisms for that transfer. (b) Crystal structures are available for three enzymes, each in at least two different states, both in the presence and absence of substrates. (c) Proton NMR spectroscopy studies have been used to examine the protein and many of its mutants, both structure and dynamics, in several states in the presence and absence of substrates. (d) There is a wealth of kinetic data on the enzyme and on mutants. These studies allow us to describe the dynamics of the reaction which can then be used in an effort to appreciate a wide range of kinetic analyses of phosphate transfer reactions.
Article
The structure of a ternary complex of the R65Q mutant of yeast 3-phosphoglycerate kinase (PGK) with magnesium 5'-adenylylimidodiphosphate (Mg-AMP-PNP) and 3-phospho-D-glycerate (3-PG) has been determined by X-ray crystallography to 2.4 angstrom resolution. The structure was solved by single isomorphous replacement, anamalous scattering, and solvent flattening and has been refined to an R-factor of 0.185, with rms deviations from ideal bond distance and angles of 0.009 angstrom and 1.78 degrees, respectively. PGK consists of two domains, with the 3-PG bound to a "basic patch" of residues from the N-terminal domain and the Mg-AMP-PNP interacting with residues from the C-terminal domain. The two ligands are separated by approximately 11 angstrom across the interdomain cleft. The model of the R65Q mutant of yeast PGK is very similar to the structures of PGK isolated from horse, pig, and Bacillus stearothermophilus (rms deviations between equivalent alpha-carbons in the individual domains < 1.0 angstrom) but exhibits substantial variations with a previously reported yeast structure (rms deviations between equivalent alpha-carbons in the individual domains of 2.9-3.2 angstrom). The most significant tertiary structural differences among the yeast R65Q, equine, porcine, and B. stearothermophilus PGK structures occur in the relative orientations of the two domains. However, the relationships between the observed conformations of PGK are inconsistent with a "hinge-bending" behavior that would close the interdomain cleft. It is proposed that the available structural and biochemical data on PGK may indicate that the basic patch primarily represents the site of anion activation and not the catalytically active binding site for 3-PG.
Article
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)
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Detailed kinetic studies, including initial velocity, product inhibition, and substrate analog inhibition measurements, have been carried out on the reverse reaction catalyzed by human erythrocyte phosphoglycerate kinase with MgATP2- as the phosphoryl group donor. The results are consistent with the reaction conforming to a rapid equilibrium random mechanism. Substantially similar results were obtained for other activating metal ions (manganese, calcium, and cobalt) and other nucleoside triphosphates (ATP, GTP). An ADP ATP exchange reaction was invariably associated with the purified enzyme but definitive evidence that it was an intrinsic property of the enzyme was not obtained.
Article
Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A 1%,1Cm 280 equals 6.7; Amax/Amin equals 2.26; e280 equals 31.5 mM(-1) X cm(-1). The enzyme consists of about 420 amino-acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino-acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half-cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S-carboxymethylated protein confirm the data of amino-acid analysis. Liver phosphoglycerate kinase is inactivated when modified with either p-chloromercuribenzoate or 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2, sugessting the presence of a disulfide bridge. Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000-50000. By analytical dodecylsulfate-polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results in the value 37500 obtained by thin-layer gel chromatography in 6 M guanidine-HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w equals 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v 0.747 ml x g(-1). The diffusion coefficient was found to be 76.9 mum2 x s(-1) by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine-liver phosphoglycerate kinase are: frictional ratio f/f0 equals 1.18, axial ratio equals 3.3, maximal degree of hydration equals 0.1 g per g of protein. Bovine-layer phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine-HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme. Some characteristics of bovine-liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.
Article
Spectrophotometric pH titrations of phosphoglycerate kinase (EC 2.7.2.3) reveal seven tyrosyl residues. In the native state one tyrosyl residue has pKapp equal to 9.3, another has pKapp of about 12.9, and five have pKapp values close to 11.0. Titration above pH 10 causes concomitant reduction of the catalytic activity. Reactivation of the enzyme occurs during storage at pH 7.8. In 6 M guanidine - HCl seven tyrosyl residues with pKapp values equal to 10.0 appear. Nitration of three tyrosyl residues occurs easily when tetranitromethane is used in excess. Four tyrosyl residues appear to be masked or buried. The tyrosyl residue having pKapp equal to 9.3 can be selectively nitrated. Simultaneously the enzyme loses 40% of its catalytic activity. No change in the Km value for one or the other of the two substrates, MgATP or 3-phospho-D-glycerate, was observed in the mononitrated enzyme. On the other hand MgATP protects the tyrosyl residue from nitration whereas 3-phospho-D-glycerate at corresponding condition appears harmless. These results suggest the low ionizing tyrosyl residue to be situated close to the binding site of MgATP, possibly in a pocket just behind. Circular dichroism measurements indicated that minor successive changes occur in the secondary structure, mainly the beta-structure, when the enzyme is being nitrated. It is reasonable to think that these structural changes, possible in combination with steric hindrance, are responsible for the decrease in catalytic activity. Dimerization of the enzyme occurs if the single thiol group is not masked before the tetranitromethane treatment.
Article
This chapter describes 3-phosphoglycerate kinase. The enzyme 3-phosphoglycerate kinase was isolated from yeast. The crystalline yeast preparation has been the principal supply of the enzyme. 3-Phosphoglycerate kinase catalyzes the transfer of “energy-rich” phosphate from the acid anhydride bond of 1,3-DPGA to the terminal phosphate of adenosine diphosphate (ADP). Divalent cations are essential to the reaction because the magnesium complexes of the nucleotides are most likely the true substrates. 3-Phosphoglycerate kinase is involved in carbon fixation in many plant tissues; it is the enzyme reacting immediately after one of the primary carbon fixing enzymes, ribulosediphosphate carboxylase. 3-Phosphoglycerate kinase has been shown to occur in a variety of higher plants and in blood, but it exists in highest concentrations in yeast, about 2 mg/g wet weight. Because of compartmentalization within the cells, it probably exists at concentrations up to four times greater than these that is up to 0.1 mM. In this chapter, biological behavior of phosphoglycerate kinase is described. Isolation and molecular properties are explained. Reaction kinetics is also discussed in the chapter.
Article
Three electrophoretic components of phosphoglycerate kinase have been isolated from baker's yeast. The isoionic point of the major component is 7.18 at 10 degrees C. Corresponding values for the minor ones are 6.91 and 7.48, respectively. There is a difference of one charge-unit between the isomers 1 and 2, and between the isomers 2 and 3. The release of component 3 from the yeast cells appears in contrast to the isomers 1 and 2 to be promoted by an organic solvent, thus suggesting this component to be bound to the cell-membrane. The amino-terminal amino acid residue appears to be N-acetylated serine in each of the three cases. The carboxyl-terminal ends seem to be identical also with -(Ala, Leu, Val, Lys)- Ala-Lys as the ultimate sequence. From the circular dichroism spectra the contents of alpha-helix and beta-structure were estimated to 15 and 40-50%, respectively. Factors have been determined for transformation and comparison of the specific activities as determined under the various conditions used at different laboratories.
Article
Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A1%, 1cm280= 6.7; Amax/Amin= 2.26; ɛ280= 31.5 mM⁻¹× cm⁻¹). The enzyme consists of about 420 amino-acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino-acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half-cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S-carboxymethylated protein confirm the data of amino-acid analysis.
Article
A reproducible procedure for the large-scale preparation of phosphoglycerate kinase frombaker's yeast is described. This method includes autolysis of dried yeast in 0.75 m ammonia, heat treatment, ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, Cibacron blue 3 G-A-Sepharose 4B pseudoaffinity chromatography, and Sephadex G-100 gel filtration. Approximately 1.7 g of homogeneous phosphoglycerate kinase can be obtained from 1 kg of air-dried bakers' yeast (yield 52%, specific activity 890 units/mg at 25°C). In a few cases further purification was achieved by reversible salting out on Sepharose CL-4B, hydroxylapatite chromatography, or ATP-Sepharose 4B affinity chromatography. Differences in the preparation of phosphoglycerate kinase from yeast with those from pig liver and pig muscle are discussed, especially concerning the interaction of the three enzymes with the chromophores of Cibacron blue- and dextran blue-Sepharose.
Article
Crystals of yeast phosphoglycerate kinase and one of its mercury derivatives have been prepared and shown to be ideally suited for further X-ray diffraction studies on this enzyme.
Article
Full-text available
An alternative method of purifying 3-phosphoglycerate kinase (EC 2.7.2.3) from yeast is described which employs step-induced pH-gradient elution from phosphocellulose and electrofocusing. The purified enzyme has a specific activity of 1200 to 1400 units per mg when corrected to saturating conditions and 30°. Reaction of this enzyme with Woodward's reagent K. (N-ethyl-5-phenylisoxazolium-3'-sulfonate) leads to rapid inhibition; MgATP affords protection against inactivation. Inhibition obeys pseudo-first order kinetics and there is an enzyme inhibitor complex formed prior to covalent modification. Although up to 5 moles of carboxyl per mole of phosphoglycerate kinase can be modified by Woodward's reagent K, it appears that only a single carboxyl is essential for the enzyme's action. The essential carboxyl appears to be more reactive than the nonessential but modifiable carboxyls. Sedimentation velocity and optical rotatory dispersion studies indicate that there has been no major conformational change concomitant with modification. Possible mechanistic roles for the essential carboxyl are discussed.
Article
Basic features of phosphoglycerate kinase (PGK), The sequences and crystal structures of PGK NMR studies of the structure of the protein PGK, Thermal analysis of yeast PGK, Additional data from NMR studies Substrate binding, Nucleotide binding site: MgATP and MgADP, The function of magnesium, The triose substrate binding site, NMR analysis of substrate binding: summary, General anion-binding site The conformation change, Kinetic studies of the reaction, Site-specific mutagenesis studies Effects of mutations on protein structure in the absence of substrates, Mutations in the interdo-main region, Mutations in the basic patch Effect of Gri3P binding on mutant proteins, Mutations in the interdomain region, Mutations in the basic patch Effects of mutations on kinetics of PGK, Mutations in the interdomain region, Mutations in the basic patch Kinetic effects of low sulphate concentration, Interdomain region, Basic patch region The so-called hinge of proteins, Features of PGK Common features with other NTP-utilising systems, The nucleotide-binding site, The catalytic loop, Domain closure Comments on mechanical devices in multi-protein enzymes
Product inhibiton studies on yeast phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) have been performed with . The results indicate that: 1.1. The catalytic reaction can be affected via four substrate binding sites, two for MgATP2− and two for .2.2. There is one catalytic centre per enzyme molecule.3.3. The catalytic reaction primarily occurs at the ‘first’ or ‘high affinity’ MgATP2− and binding sites. The ‘second’ set of sub-sites for these substrates are located in a region for regulatin of the catalytic reaction.4.4. The products of the reaction, and ADP, are preferentially bound to the regulatory region.5.5. MgATP2− and are able to bind simultaneously to this region. When liganded with MgATP2− the apparent value for increases from 3 μM to 20 μM.
Article
Full-text available
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.
Article
The adenovirus-type-5-coded single-strand-specific DNA-binding protein was purified from infected human KB cells and characterized by sucrose gradient centrifugation and gel filtration. The protein has a sedimentation coefficient of 3.3 S and a diffusion coefficient of 4.4 × 10−7 cm/s, which corresponds to a molecular weight of 68000 for the native protein. This is close to the peptide molecular weight of 72000 and indicates that the protein is a monomer. The frictional coefficient ratio is 1.82 which suggests that the DNA-binding protein is highly anisometric. The binding of the protein to various nucleic acids was studied by filtration through nitrocellulose filters and sucrose gradient centrifugation. The binding to single-stranded DNA is a fast process which does not require bivalent cations or sulfhydryl groups and occurs over a broad pH range. The binding to synthetic polydeoxyribonucleotides is at least 10-fold less efficient than that to natural single-stranded DNA. At low protein-to-DNA ratios the binding to single-stranded DNA is cooperative. A maximal protein to DNA ratio of 33 (w/w) can be obtained which corresponds to 1 protein molecule for about 7 nucleotides. Sedimentation and electron microscopic studies indicate that the DNA-binding protein keeps single-stranded DNA in an extended configuration. The saturated nucleoprotein complex is slightly more resistant to nuclease digestion than single-stranded DNA.
Chapter
This chapter describes that enzymes are globular proteins with spherical or ellipsoidal molecular shapes. Their structures are suitable for catalyzing various degradative, synthetic, and transformations reactions in biological systems. Compared with other proteins, enzyme proteins have some characteristic structural features that may be important for their electrophoretic separation and detection. The compactness of enzymic proteins leaves little room for water molecules inside the protein molecule. Many enzymes that contain more than one polypeptide are called “oligomeric,” and can be dissociated into functional or regulatory subunits. Enzymes consisting of more than one type of subunit can produce isoenzymes because of variations in the combination of the subunits in the protein molecule. Enzymes involved in oxidation–reduction reactions, contain non-amino acid components, which can be organic or inorganic, or both. The International Union of Biochemistry (IUB) divides all enzymes into six major classes and several subclasses, according to the type of reaction catalyzed.
Article
Reaction of yeast phosphoglycerate kinase with either butanedione or cyclohexanedione can result in modification of up to all 13 arginyl residues with total loss of activity; however, extrapolation to zero activity for partially modified preparations indicates that up to 7 arginyls are essential. Whereas 20 mm 3-phosphoglycerate affords partial protection of activity toward both reagents, 20 mm MgATP affords complete protection of activity and protects 2 arginyls against modification by butanedione and 1 arginyl against modification by cyclohexanedione. With butanedione the modification could be reversed with total recovery of activity, suggesting that only arginyl groups were modified, which is consistent with the amino acid analysis of the modified protein. Only at high cyclohexanedione concentrations or long reaction times was a yellow product obtained that showed loss of lysyl residues. Circular dichroism spectra show that even when all the arginyls are modified by butanedione or up to 10 modified by cyclohexanedione there is no change observed in the far or near ultraviolet, indicating that there is no detectable conformational change concomitant with modification, which is confirmed by hydrodynamic studies. It is concluded that at least one, possibly two, arginyls of yeast phosphoglycerate kinase are essential for its action on ATP.
Article
The effects of urea in concentrations from 0 to 6M on the following properties of yeast phosphoglycerate kinase were studied: the kinetics of inactivation of the enzyme, the spectrum of 2-chloromercuri-4-nitrophenol bound to the single thiol group of the enzyme, the rate of reaction between the mercurial and enzyme, and the isoelectric point. The enzyme was inactivated by as much as 30% in 1M-urea, and the other data were interpreted as a possible 'tightening' of enzyme structure. The catalytic behaviour of the enzyme in 2M-urea was time-dependent, the initial effects being similar to those in 1M-urea. Polyacrylamide-gel isoelectric focusing of the enzyme in the presence of 2M-urea showed a single species of enzyme with an isoelectric point intermediate between those in 1M- and 3M-urea; a species with an identical isoelectric point was obtained after an 11-day exposure at 4 degrees C to the denaturant at 2M. The enzyme was rapidly inactivated in 3M-urea, with the thiol group fully exposed and the isoelectric point 0.9pH unit higher than in the absence of urea. No further conformational changes could be demonstrated with urea concentrations of 4M or greater. It is suggested that the equilibrium species that exists in 2M-urea has one of two buried lysine residues exposed. The second lysine residue is exposed in 3M or greater concentrations of the denaturant.
Article
A reproducible procedure for the large-scale preparation of phosphoglycerate kinase frombaker's yeast is described. This method includes autolysis of dried yeast in 0.75 m ammonia, heat treatment, ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, Cibacron blue 3 G-A-Sepharose 4B pseudoaffinity chromatography, and Sephadex G-100 gel filtration. Approximately 1.7 g of homogeneous phosphoglycerate kinase can be obtained from 1 kg of air-dried bakers' yeast (yield 52%, specific activity 890 units/mg at 25°C). In a few cases further purification was achieved by reversible salting out on Sepharose CL-4B, hydroxylapatite chromatography, or ATP-Sepharose 4B affinity chromatography. Differences in the preparation of phosphoglycerate kinase from yeast with those from pig liver and pig muscle are discussed, especially concerning the interaction of the three enzymes with the chromophores of Cibacron blue- and dextran blue-Sepharose.
Article
We have critically evaluated hydrodynamic data from 21 proteins whose molecular dimensions are known from X-ray crystallography. We present two useful equations relating the molecular weights and sedimentation coefficients of globular proteins. The hydrodynamic data combined with data for small molecules from the literature indicate that failure of the Stokes equation occurs only for molecular weights <850. Calculated hydration values for the 21 proteins have a mean value and standard deviation of 0.53 ± 0.26 g H2O/g protein. Furthermore, statistical arguments indicate that only 5.3% of the variance is due to experimental error. The mean value and especially the dispersion of values are in sharp contrast to the values 0.36 ± 0.04 obtained by others from nmr measurements on frozen protein solutions. Hydration values calculated from nmr measurements are closely correlated with the number of charged and polar amino acid residues. In contrast to this result, our analysis of the amino acid compositions of the four proteins with the lowest hydration and the four monomeric proteins with the highest shows that the range of values we observe cannot be accounted for on the basis of amino acid composition. In fact there appears to be a weak correlation between the number of apolar residues and hydrodynamic hydration. We therefore conclude that the dispersion must result from variations in fine details of the surface structures of individual proteins. We propose a model of hemispherical clathrate cages which if correct, would account for the differences in the data obtained by these two methods.
Article
The kinetics of slow phase reactivation of 11 single chain denatured enzymes containing between 6 and 28 proline residues were each found to be first-order having half-times ranging from 0.15 to 12.1 minutes, respectively, at 25 °C. The reactivation kinetics of selected enzymes are independent of solvent viscosity and give an activation energy of 19 kcal/mol. These results are consistent with the proposal that cis/trans proline isomerization in the denatured state is responsible for the slow phase of enzyme refolding/reactivation and with biosynthetic rates for enzyme production.
Article
1. A mol.wt. of 40030 +/- 830 has been estimated for phosphoglycerate kinase in concentrations less than 0.1 g/100 cm3 comparing favourably with expected values from X-ray diffraction measurements by 10% lower than the previously reported molecular weights made at higher concentrations. 2. The so20w, was estimated to be 3.12(+/-0.02)x10(-13)s and the coefficient had a low concentration dependency giving a g value (concentration-dependency) of 2.3 +/- 1.6cm3 .g-1. This agrees with previous qualitative observations. 3. By using fluctuation-intensity spectroscopy, the D20,w was estimated to be 7.4(+/-0.2)x10(-11)m2.s-1, and this was indistinguishable from the D20,w calculated from ultracentrifuge results. The water of hydration was estimated to be 0.46 g/g of protein. 4. It is inferred from the estimates that phosphoglycerate kinase associates with an interaction coefficient at 20 degrees C for monomer/dimer of between 10 and 12 cm3.g-1. 5. The ratio of molecular asymmetry (a/b) was estimated to be 2.5+/-0.2 from the values of D20,w and water of hydration. This compares favourably with the ratio from the overall dimensions estimated from X-ray diffraction measurements.
Article
Phosphoglycerate kinase (ATP:3-phospho-D-glycerate-1-phosphotransferase, EC 2.7.2.3) from young and old Turbatrix aceti has been purified to homogeneity. The "old" enzyme exhibits a marked reduction in specific activity both in crude homogenates and in pure form when compared to preparations from young nematodes. The specific activities for pure "young" and "old" enzymes are 650-750 and 300-400 units/mg, respectively. All other properties of "young" and "old" enzymes were nearly identical, including molecular weight (43 000), Km, behavior on columns, thermal stability and mobility during gel electrophoresis at three pH values. The results are discussed in terms of the possible mechanism of formation of "altered" enzymes. In addition, certain properteis of the nematode phosphoglycerate kinase are compared with those of the enzyme from yeast and rabbit muscle.
Article
Full-text available
Sulfhydryl reagents, as well as mild hydrogen peroxide oxidation, do not inhibit the activity of yeast phosphoglycerate kinase, indicating that the single thiol group and 3 methionine residues present in the enzyme are not essential for activity. Nitration of phosphoglycerate kinase by tetranitromethane inhibits the enzyme by reaction with a single tyrosine residue. Substrates provide partial protection against inactivation by nitration. Circular dichroism spectra indicate that no conformational changes occur upon nitration. However, perturbation of the microenvironment surrounding the aromatic amino acid residues, particularly tyrosine, was observed. The same perturbation was observed on addition of the substrate 3-phosphoglycerate kinase to native phosphoglycerate kinase. The role of lysine in the action of yeast phosphoglycerate kinase has been studied by modification with O-methylisourea, 2-methoxy-5-nitrotropone, and pyridoxal phosphate. Guanidination shows that there are lysines essential for phosphoglycerate kinase; extrapolation to zero activity indicates that there are three essential lysines as judged by nitrotroponylation and three essential lysines when the enzyme is reacted with pyridoxal phosphate. Substrates afford partial protection and extrapolation to total protection indicates that up to three lysines are protected by MgITP and one lysine by 3-phosphoglycerate. Spectrofluorescence and optical rotatory dispersion measurements show that there is no detectable conformational change for the guanidinated phosphoglycerate kinase and that there are slight changes in the spectra suggesting that there may be slight conformational changes for the nitrotroponylated and the pyridoxal phosphate-modified enzymes.
Chapter
This chapter reviews that the simplest way of accounting for the absorption spectrum of a protein is as the sum of the spectra of its components. This gives results which are often good approximations to the observed protein spectrum. The assumption of additivity is basic for useful analytical applications of spectral measurements. It discusses that in another perspective, the failure of perfect additivity of the component absorptivities affords the possibility of obtaining structural information about proteins. The absorptivity of the peptide bond can change by as much as a factor of two with a change in the conformation of a peptide chain. Since the absorptivity of aromatic side chains is much less sensitive to environmental change, correspondingly, more sensitive techniques are required to measure the small changes which do occur. The nature and limitations of the structural information resulting from both peptide-bond and side-chain absorption are discussed. Absorption spectra can occasionally be a useful tool in the identification of unusual structural features in proteins and polypeptides. Several such applications are also explained in this chapter.
1.|The Michaelis constants for the two substrates of phosphoglycerate kinase (ATP:d-3-phosphoglycerate i-phosphotransferase, EC 2.7.2.3), MgATP2− and 3-phosphoglycerate, are each independent of the concentration of the second substrate. Three prevalent mechanisms can describe this situation.
Article
1.1. The substrate kinetics of the enolase reaction was studied. High substrate concentrations were shown to have an inhibitory effect. Possible reaction mechanisms were discussed.2.2. The influence of the concentration of Mn++ and Zn++ on enolase activity was investigated. The activity-concentration curves were found to go through a maximum. An equation describing this effect was derived by assuming that the metal ions interact with two sets of sites on the enolase molecule, one leading to activation and the other to inhibition.3.3. The maximum activity with a given concentration of enzyme was not the same for the different metal ions. It was suggested that the ionic radius of a given metal ion is the main property determining its activating power.4.4. The interaction of enolase with Mn++ and Zn++ was studied by equilibrium dialysis. By comparison with the activation experiments, it was concluded that the activation involves the binding of one single metal ion per molecule of enzyme. Additional metal ions were found to be bound, but this resulted in inhibition.
Article
Improved procedures have been developed for the chromatographic determination of amino acids on columns of finely pulverized 8 % cross-linked sulfonated polystyrene resins. The use of a smaller particle size has permitted faster flow rates, and appropriate choice of eluents has simplified operations. Complete analyses of protein hydrolyzates can be performed in about 48 hours with fraction collectors and in 24 hours with automatic recording equipment. The same system, with minor modifications, can be used for determination of amino acids and related compounds in blood plasma, urine, and animal tissues.
Article
The binding of Mn++ to 3-phosphoglycerate kinase, and to ADP and ATP, has been studied at pH 7.1 and 38 °. The influence of the concentrations of Mn++ and of the various substrates on the rate of the enzymic reaction has also been investigated at the same conditions. The protein displays only a weak, nonspecific interaction with Mn++. By correlating the kinetic and binding data, it is possible to show that activation does not involve binding of Mn++ to the enzyme but rather the formation of a Mn++-ATP complex. The significance of the results in relation to the general problem of the mechanism of metal-ion activation of enzymes is discussed.
Article
1.1. Apparatus for zone electrophoresis in columns has been described.2.2. Directions for packing and operation of the columns have been given.3.3. The behavior of some compounds of low molecular weight on cellulose columns under different conditions has been studied. Thus adsorption and zone-spreading as well as temperature rise within columns of different diameters as a function of current have been investigated.4.4. the usefulness of zone electrophoresis in cellulose columns for fractionating mixtures of amino acids and related compounds, pea root exudate and crude pituitary extract has been demonstrated.
Article
1.1.|With an injection technique, glycolytic oscillation can be induced in yeast extract with hexoses, glucose-6-phosphate, fructose-6-phosphate, but not fructose-1,6-diphosphate, with an average rate corresponding to a QNglucose of 70 (per intact cell).2.2.|The enzyme and metabolic concentration pattern of glycolysis of a yeast extract is presented. Enzyme molarities per cytosol in the order of 10−6 to 10−4 are found. The enzyme activity pattern for optimal and oscillating conditions reveals large amplitude oscillations (∼ 60-fold) of the activity changes of phosphofructokinase during the oscillation and a mean activity of all glycolytic enzymes in the range of 10–40% of the optimal activity.3.3.|Glycolytic self-excitation is induced by substrate addition and generated by the allosteric oscillophor phosphofructokinase. The pulsed generation of products by phosphofructokinase is propagated with a dependent phase shift by means of the coupling variable adenosine phosphate via the kinases, phosphoglycerate kinase and pyruvate kinase. The function of fructose-diphosphate during the oscillations is masked because oscillations only occur above the stresshold level of fructose-diphosphate being critical for the activation of pyruvate kinase. The function of ATP and ADP is amplified by the strong activation of phosphofructokinase by AMP.4.4.|Oscillations are a general property of metabolic systems and an implicit function of their feedback structure, which involves cross-coupling and self-coupling with opposite sign in a two variable structure and might produce kinetic instability involving more than one singularity of the trajectories in a phase plane. The study of oscillations reveals the dynamics of a pathway over a large range of states. The physiological significance of controlled oscillations can not yet be evaluated.
Article
1. A procedure for preparing crystalline 3-phosphoglycerate kinase from rabbit or pig skeletal muscle is presented. 2. The preparation phosphorylates up to 975mumoles of 3-phosphoglycerate/min./mg. at 30 degrees and is not contaminated with myokinase. 3. The enzyme has an estimated molecular weight of 36500+/-1000, and contains three residues each of tyrosine and tryptophan. 4. The preparation is suitable for use in the enzymic procedures for determining ATP, phosphocreatine and 3-phosphoglycerate.
Article
1.1.|The Michaelis constants for the two substrates of phosphoglycerate kinase (ATP:d-3-phosphoglycerate i-phosphotransferase, EC 2.7.2.3), MgATP2− and 3-phosphoglycerate, are each independent of the concentration of the second substrate. Three prevalent mechanisms can describe this situation.2.2.|The Mg2+ complex of 3-phosphoglycerate does not appear to be in an active substrate form.3.3.|Mg2+ at high concentrations inhibits the enzyme non-competitively with respect to 3-phosphoglycerate.4.4.|High concentrations of Mg2+ change the kinetic relationships and non-linear Lineweaver-Burk plots are obtained for the two substrates. The curves can be approximated to two straight lines. The two intersection points with the abciss axis are independent of the concentration of the second substrate.5.5.|The data are interpreted in terms of two different binding sites for each substrate, the second set of sites being involved only at high Mg2+ concentrations. Various mechanisms for this effect are considered and it is suggested that these could also be important for other enzymes involving reaction with ATP.
Article
1. 1. The substrate kinetics of the enolase reaction was studied. High substrate concentrations were shown to have an inhibitory effect. Possible reaction mechanisms were discussed. 2. 2. The influence of the concentration of Mn++ and Zn++ on enolase activity was investigated. The activity-concentration curves were found to go through a maximum. An equation describing this effect was derived by assuming that the metal ions interact with two sets of sites on the enolase molecule, one leading to activation and the other to inhibition. 3. 3. The maximum activity with a given concentration of enzyme was not the same for the different metal ions. It was suggested that the ionic radius of a given metal ion is the main property determining its activating power. 4. 4. The interaction of enolase with Mn++ and Zn++ was studied by equilibrium dialysis. By comparison with the activation experiments, it was concluded that the activation involves the binding of one single metal ion per molecule of enzyme. Additional metal ions were found to be bound, but this resulted in inhibition.
Article
The binding of Mn++ to 3-phosphoglycerate kinase, and to ADP and ATP, has been studied at pH 7.1 and 38 °. The influence of the concentrations of Mn++ and of the various substrates on the rate of the enzymic reaction has also been investigated at the same conditions. The protein displays only a weak, nonspecific interaction with Mn++. By correlating the kinetic and binding data, it is possible to show that activation does not involve binding of Mn++ to the enzyme but rather the formation of a Mn++-ATP complex. The significance of the results in relation to the general problem of the mechanism of metal-ion activation of enzymes is discussed.
Article
The effect of the concentration of Mg2+, ATP4- and Mg-ATP2- on the velocity of the phopshoglycerate kinase (ATP:d-3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3) reaction has been studied. Partly new applications of known kinetic equations are given. Three different kinetic relationships support the concept that Mg-ATP2- is the active substrate. It is also seen that mechanisms in which the formation of enzyme-metal or enzyme-nucleotide complexes are the first steps in the catalytic reaction are very unlikely. ATP4- and Mg2+ both seem to have inhibitory effects only.
Article
1.1. Amino acid analyses and determinations of elementary composition of various forms of bovine and human erythrocyte carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) have been carried out. A technique for the calculation of molecular weights from amino acid composition is described.2.2. A feature common to all of the forms of the enzyme studied is their low content of sulfur-containing amino acids which excludes the occurence of disulfide bridges in the molecules. The bovine enzyme contains no cysteine and it is thus possible to exclude a sulfhydryl group as a ligand in the binding of the zinc ion to the protein.3.3. Great differences in amino acid composition are found between forms from the different species as well as between different forms of the human enzyme. However, some of the electrophoretically separable forms of the enzyme show identical amino acid composition within the experimental errors. The possible biosynthetic relations between the forms of the human enzyme are discussed.
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