Yeast Pyruvate Kinase Research Articles

The effect of pH on the interaction of substrates and effector to yeast and rabbit muscle pyruvate kinase

The interaction of fructose 1,6-bisphosphate, phospho enolpyruvate and ADP with pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) from yeast and rabbit muscle has been studied as a function of pH utilizing the quenching of protein fluorescence at 330 nm by these ligands. Both the muscle and the yeast pyruvate kinase interact with either ADP or phospho enolpyruvate with similar affinity, indicating that the substrate-binding sites for these two isozymes are similar. The major difference between the yeast and muscle isozymes is their affinity with fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate interacts with the yeast isozyme in orders of magnitude more strongly than with the muscle isozyme. Moreover, the affinity of fructose 1,6-bisphosphate to the yeast isozyme is strongly pH-dependent, while the interaction of fructose 1,6-bisphosphate with the muscle isozyme is independent of pH. The data indicate that yeast pyruvate kinase undergoes a conformational change as the pH is increased from 6.0 to 8.5.

Yeast pyruvate kinase catalyzes the hydrolysis of phospho enolpyruvate in the absence of ADP

In the absence of ADP as phosphoryl acceptor, yeast pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) catalyzes the hydrolysis of phospho enolpyruvate. The reaction depends linearly on enzyme concentration, requires magnesium ions, which cannot be replaced by calcium ions, and is allosterically activated by fructose 1,6-bisphosphate, as is the physiological reaction. The hydrolysis has a specific activity of 6.2 · 10 −4 μmol/min per mg and exerts positive cooperativity ( n H = 2.0). The activation by fructose 1,6-bisphosphate is allosterically affected by phospho enolpyruvate. It is suggested that in the rate-limiting step a magnesium ion directly activates the phosphoryl group and that a metal coordinated water molecule protonates the enol-oxygen. The initial product enolpyruvate is then ketonized at the active site of the enzyme.

The regulatory properties of yeast pyruvate kinase.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.

A new approach to understanding kinetic cooperativity when the Hill coefficients are less than 2

The simple enzyme kinetic equation, V ̄ v = 1 + K [ S] + D (A + [ S]) , allows one to obtain more information than does the Michaelis-Menten equation, from many nonlinear Lineweaver-Burk double-reciprocal plots in terms of four useful empirical parameters V ̄ , K, D, and A . Thus, in the absence of substrate inhibition, maximum or minimum Hill coefficients ( H) from 0 to 2 are given by the expression H 2 (1 − H) = 4K D . Only the parameter D can be negative and this is associated either with positive kinetic cooperativity (1 < H < 2) or substrate inhibition (( K + D) ≤, 0). By assuming that dissociation of the product is an exchange reaction coupled with either binding of the substrate or a surrogate ligand (competitive inhibitor), a minimal model was obtained that defines these four empirical parameters in terms of only five specific chemical reaction rate constants that can, in fact, be evaluated experimentally. In this model negative kinetic cooperativity (0 < H < 1) results when the substrate displaces the modifier more easily than the product and positive kinetic cooperativity (1 < H < 2) when the converse is true, whether this occurs by a direct displacement or through allosteric interactions. This simple minimal model successfully predicts how the parameters vàry with the concentration of surrogate ligand (competitive inhibitor) in several instances. In particular a new equation [ 1 (H − 1) = α + β[ modifier]] that relates the Hill coefficient ( H) to the modifier concentration was derived and shown to be empirically valid for both Eschericia coli phosphofructokinase and yeast pyruvate kinase even though they have Hill coefficients up to 4.

Affinity labeling of the active site of yeast pyruvate kinase by 5'-p-fluorosulfonylbenzoyl adenosine.

Yeast pyruvate kinase is irreversibly inactivated by 1.1 mM 5'-p-fluorosulfonylbenzoyl adenosine at pH 8.6 with an initial rate constant of 0.019 min-1. A plot of kinact versus the 5'-p-fluorosulfonylbenzoyl adenosine concentration yields a hyperbolic curve indicative of binding of the analog prior to reaction. Marked protection is afforded by phosphoenolpyruvate + fructose 1,6-diphosphate + Mg2+ or MgATP suggesting that reaction occurs within the active site. When assayed at less than saturating phosphoenolpyruvate concentrations, the inactivation caused by the reagent in the absence of added ligands appears slower, and reaction in the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+ produces an activation of the enzyme, the extent of which is dependent on the assay concentration of phosphoenolpyruvate. The rate constant for activation was observed to be 0.113 min-1. The activated enzyme exhibits both a lowered K0.5 and Hill coefficient compared to native pyruvate kinase. Subsequent addition of 5'-p-fluorosulfonylbenzoyl adenosine to activated pyruvate kinase in the absence of added ligands leads to inactivation with the rate constant independent of the assay concentration of phosphoenolpyruvate. Covalent reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyl adenosine thus occurs at two distinct sites. In the presence of phosphoenolpyruvate, fructose 1,6-diphosphate, and Mg2+, incorporation of tritiated 5'-p-fluorosulfonylbenzoyl adenosine is linearly proportional to the extent of activation of the enzyme, with 4 mol of reagent bound/mol of tetrameric pyruvate kinase for maximally activated enzyme. In the absence of added ligands, approximately 4.5 mol of reagent are incorporated/mol of enzyme at 15 min of reaction, while 80% of the original activity remains. Subsequent incorporation is proportional to the extent of inactivation with 8 mol bound at 100% in activaton. In the presence of phosphoenolpyruvate, fructose 1,6-diphospate, and Mg2+, 3 tyrosines and 1 lysine residue, and in the absence of ligands, 6 tyrosines and 2 lysine residues are modified, suggesting that both amino acids are within the two nucleotide sites.

Comparative studies of kinetic and optical properties of rabbit muscle, sturgeon muscle, and yeast pyruvate kinase

The kinetic and optical properties of pyruvate kinase isolated from rabbit muscle, sturgeon muscle, and yeast were compared using various activating divalent metal ions as probes for functional features and using ultraviolet circular dichroism (cd) measurements for conformational features, respectively. All three preparations of pyruvate kinase were similar in many aspects, such as activating efficiencies of the four activating metal ions, Mg(II), Co(II), Mn(II), and Ni(II) and pH-rate profiles, suggesting the presence of a similar metal binding locus of these enzymes as well as a common underlying mechanism of action. L-Phe inhibited the rabbit muscle enzyme and turned the hyperbolic kinetics into a sigmoidal kinetic with respect to phosphoenolpyruvate at alkaline pH, while fructose-1,6-biphosphate activated the sturgeon muscle and yeast enzymes and turned the sigmoidal kinetics into hyperbolic kinetics with respect to phosphoenolpyruvate. The ultraviolet cd spectral changes qualitatively correlated well with kinetic observations of all three native enzymes in the presence and absence of allosteric effectors. Our results suggested that there are at least two conformational states of pyruvate kinase which are inducible by the binding of substrate and (or) allosteric effectors. The conformational changes from one form to another in these enzymes are very similar, especially between the rabbit and sturgeon muscle enzymes.

Kinetics study of yeast pyruvate kinase after modification of exposed sulfhydryl residues.

Kinetics study of yeast pyruvate kinase after modification of exposed sulfhydryl residues.

Modification of yeast pyruvate kinase by an active site-directed reagent, bromopyruvate.

Modification of yeast pyruvate kinase by an active site-directed reagent, bromopyruvate.

Studies on the macroconidia of Microsporum canis

The characteristics of an in vitro polyuridylic acid dependent amino acid incorporating system prepared from germinating macroconidia of Microsporum canis are described. The incorporation of 14C-phenylalanine into polyphenylalanine is dependent on S-30 extract, adenosine triphosphate, magnesium ions and polyuridylic acid. Incorporation is slightly enhanced by yeast transfer ribonucleic acid and pyruvate kinase. The system is highly sensitive to ribonuclease, puromycin and miconazole (an antifungal agent), moderately sensitive to sodium fluoride and much less sensitive to phenethylalcohol, cycloheximide, chloramphenicol and deoxyribonuclease. Cell-free extract from ungerminated conidia has less capacity to synthesize the protein and during germination a marked increase in the protein synthetic activity is observed. The results from experiments wherein ribosomes and S-100 fraction from germinated and ungerminated spores are unterchanged, revealed that the defect in the extract from the ungerminated spore is in the ribosomes.

Yeast pyruvate kinase: a mutant from catalytically insensitive to fructose 1,6-bisphosphate.

The paper describes some of the characteristic properties of an altered form of pyruvate kinase from a mutant of Saccharomyces cerevisiae. The partially purified enzyme does not require fructose 1,6-bisphosphate for activity but is stabilised in its presence both at low and at high temperatures. The enzyme displays in the absence of fructose 1,6-bisphosphate hyperbolic kinetics with phosphoenolpyruvate (Km, 0.11 mM), ADP (Km, 0.12 mM) and K+ (Km, 11 mM). Sedimentation velocity experiments indicate that the mutated enzyme and the wild type enzyme have S20,w values of 8.9 and 8.6 S respectively. The mutant with the pyruvate insensitive to fructose 1.6-bisphosphate is capable of growing on synthetic media with alcohol or malate as the sole carbon source. The steady-state intracellular levels of phosphoenolpyruvate in the mutant suggest mechanisms that prevent depletion of this metabolite despite an active pyruvate kinase. Spontaneous reversion of this mutant yields clones with normal enzyme activated by fructose 1,6-bisphosphate.

The proton transfer reactions catalyzed by yeast pyruvate kinase

1. 1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10–100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction of pyruvate kinase. 2. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase (Robinson, J.L. and Rose, I.A. (1972) J. Biol. Chem. 247, 1096–1105). The pH-profiles and the divalent cation dependencies were similar for Fru1,6- P 2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization by yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization by the muscle enzyme which proceeds when ATP is replaced by P i or other dianions. 3. 3. Fructose-1,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme.

Secondary kinase reactions catalyzed by yeast pyruvate kinase

1. 1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. With Mg 2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-bisphosphate. With Mn 2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. 3. The effect of other divalent cations and pH on the three secondary kinase reactions was also examined. 4. 4. Results are compared with those obtained for muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.

Tautomeric and anomeric specificity of allosteric activation of yeast pyruvate kinase by D-fructose 1,6-bisphosphate and its relevance in D-glucose catabolism

Tautomeric and anomeric specificity of allosteric activation of yeast pyruvate kinase by D-fructose 1,6-bisphosphate and its relevance in D-glucose catabolism

Anomeric specificity of yeast pyruvate kinase toward activation by D-fructose 1,6-bisphosphate

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAnomeric specificity of yeast pyruvate kinase toward activation by D-fructose 1,6-bisphosphateRichard Fishbein, Patricia A. Benkovic, and Stephen J. BenkovicCite this: Biochemistry 1975, 14, 18, 4060–4063Publication Date (Print):September 1, 1975Publication History Published online1 May 2002Published inissue 1 September 1975https://doi.org/10.1021/bi00689a021RIGHTS & PERMISSIONSArticle Views48Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (436 KB) Get e-Alerts Get e-Alerts

Difference in pyruvate kinase regulation among three groups of yeasts

Yeast pyruvate kinase (ATP : pyruvate 2- O-phosphotransferase EC 2.7.1.40) was classified into three groups based on the interaction with fructose-1,6-bisphosphate. The pyruvate kinases of Saccharomyces cerevisiae and Saccharomyces carlsbergensis were activated by fructose 1,6-bisphosphate in the concentration range tested (up to 10 mM) of the substrate, phosphoenol-pyruvate; the enzymes of “fermentative Candida” ( Candida tropicalis and Candida utilis) were affected by fructose 1,6-biphosphate only when the substrate concentration was below 2 mM. Although the pyruvate kinase of Candida lipolytica (a yeast belonging to “oxidative Candida”) was also affected by fructose 1,6-bisphosphate, the degree of the activation was extremely small as compared with the above four yeasts. The pyruvate kinase of C. tropicalis was inhibited by ATP more strongly in the absence of fructose 1,6-bisphosphate than its presence. In the case of the C. lipolytica enzyme, however, the enzyme was inhibited to a lesser extent by ATP, and fructose 1,6-bisphosphate did not reverse the inhibitory effect of ATP. Time course changes of the enzyme levels in the yeast grown on glucose and on ethanol indicate that the pyruvate kinases of S. cerevisiae and C. tropicalis can be controlled both by an allosteric mechanism and by changes in the enzyme concentration, although a marked difference was observed in the susceptibility to the allosteric effect by fructose 1,6-biphosphate between these fermentative yeasts. On the other hand, that of C. lipolytica would be controlled only by the latter mechanism.

Chemical specificity of pyruvate kinase from yeast

Three analogs of phosphoenolpyruvic acid: (Z)-phosphoenol-3-fluoropyruvate, (Z)-phosphoenol-3-bromopyruvate and (Z)-phosphoenol-α-ketobutyrate were found to be substrates for yeast pyruvate kinase (ATP : pyruvate (Z)- O-phosphotransferase, EC 2.7.1.40) with maximal velocities much greater than those found for rabbit muscle pyruvate kinase. The analogs exhibited sigmoidal kinetics, which become hyperbolic upon addition of the allosteric effector, fructose 1,6-diphosphate. Moreover, the reaction of (Z)-phosphoenol-3-bromopyruvate with ADP to produce bromopyruvic acid and ATP irreversibly inhibited the enzyme with a half-life of 32 min.

The preparation and properties of pyruvate kinase from yeast.

A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn(2+) ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn(2+)-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein).

Structure and Function of Yeast Pyruvate Kinase

Structure and Function of Yeast Pyruvate Kinase

Modification of histidine residues in yeast pyruvate kinase by diethylpyrocarbonate.

Modification of histidine residues in yeast pyruvate kinase by diethylpyrocarbonate.

Allosteric kinetics of pyruvate kinase of Saccharomyces carlsbergensis

The allosteric model of Monod et al. (1965) has been used to analyse the steadystate kinetics of pyruvate kinase from Saccharomyces carlsbergensis. The dissociation constants for the substrate phosphoenolpyruvate, the inhibitor ATP as well as the activator fructose-1, 6-diphosphate from the R and T state were calculated using a series of computer programs. On the basis of a crucial relation (derived in the Appendix), which correlates the Hill coefficient and the half-saturating concentration of substrate saturation curves with the parameters of the model of Monod et al., it is possible to differentiate between exclusive and non-exclusive ligand binding. On the other hand, this relation makes it possible to fit the experimental data to an extended model assuming only partially concerted transitions in each enzyme molecule. The physical data of yeast pyruvate kinase point to a tetrameric structure, whereas the steady-state kinetics favour a trimeric one. This discrepancy in the number of protomers can be overcome by the use of an extended model, which permits the occurrence of hybrid states R tT n−t. The introduction of one symmetrical hybrid state R 2T 2 into the model explains the kinetic data of yeast pyruvate kinase on the basis of four, probably identical, protomers. The equilibrium constants between the states are given. In the Appendix the derivation of the equation describing the occurrence of hybrid states is reported.