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PubMed2001

Dimethylsulfoxide promotes K+-independent activity of pyruvate kinase and the acquisition of the active catalytic conformation.

Pyruvate kinase requires K+ for maximal activity; the enzyme exhibits 0.02% of maximal activity in its absence [Kayne, F. J. (1971) Arch. Biochem. Biophys. 143, 232-239]. However, pyruvate kinase entrapped in reverse micelles exhibits an important K+-independent activity [Ramírez-Silva, L., Tuena de Gómez-Puyou, M., & Gómez-Puyou, A. (1993) Biochemistry 32, 5332-5338]. It is possible that the amount of water, as well as interactions of the protein with the micelles, can account for this behavior. We therefore explored the solvent effects on the catalytic properties of muscle pyruvate kinase. The enzyme exhibited an activity of 19.4 micromol x min(-1) x mg(-1) in 40% dimethylsulfoxide, compared with 280 and 0.023 micromol x min(1) x mg(-1) observed with and without K+ in water, respectively. pH activity profiles and kinetic constants for the substrates of pyruvate kinase in dimethylsulfoxide without K+ were similar to those in 100% water with K+, and differed from those in water without K+. The spectral center of mass of the emission spectrum of pyruvate kinase in 100% water exhibited a blue shift of 3.5 nm in the presence of Mg(2+), phosphenolpyruvate, and K+, ligands that induce the active conformation of the enzyme. The spectral center of mass of the apoenzyme in 30-40% dimethylsulfoxide coincided with that of the enzyme-Mg(2+)-phosphenolpyruvate-K+ complex in 100% water. The water relaxation rate enhancement factor and binding of phosphenolpyruvate to the pyruvate kinase-Mn(2+)-(CH3)4N+ complex in 30-40% dimethylsulfoxide were similar to those of the pyruvate kinase-Mn(2+)-K+ complex in water. The aforementioned results indicate that when muscle pyruvate kinase is without K+, 30-40% dimethylsulfoxide induces its active conformation.

Binding Sites↗
PubMed1973

Pyruvate kinase, a possible regulatory enzyme in higher plants.

A number of plant species were examined for the presence of pyruvate kinase (pyruvate-ATP phosphotransferase, EC 2.7.1.40), and of a phosphatase activity which hydrolyzes phosphoenolpyruvate. Of those examined, only cotton (Gossypium sp. L.) seeds were found to be sufficiently free of the phosphatase to permit a kinetic study of pyruvate kinase.During germination of cotton seeds, pyruvate kinase activity rises for the first 3 days, after which it falls back to its original level. This developmental pattern is characteristic of enzymes involved in the conversion of fat into carbohydrate in fatstoring seeds. The phosphatase also rose rapidly during germination, which precluded the use of extracts from seedlings in the study of pyruvate kinase. No evidence was found for the presence of more than one pyruvate kinase in cotton seedlings.In crude extracts from ungerminated seeds, the enzyme shows slight deviations from normal kinetics with respect to phosphoenolpyruvate, magnesium, and to a lesser extent, ADP. After partial purification of the enzyme by ion exchange chromatography, the enzyme shows normal kinetics. The enzyme is activated by AMP, and inhibited by both ATP and citrate, in both crude and partially purified preparations. It is suggested that cotton seed pyruvate kinase is a regulatory enzyme.

Journal Article↗
PubMed1976

Hormonal control of pyruvate kinase activity and of gluconeogenesis in isolated hepatocytes.

Treatment of isolated rat hepatocytes with saturating concentrations of glucagon caused several modifications properties of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40): S0.5 (substrate concentration at half maximum velocity) for phosphoenolpyruvate was about doubled, whereas Vmax was not changed; the activity measured at 0.15 mM phosphoenolpyruvate (physiological concentration) was reduced 65-80%; and there was also an increase in the Hill coefficient and in the affinity of the enzyme for the inhibitors Mg-ATP and alanine. Glucagon, 3':5'-cyclic AMP, and epinephrine caused an inactivation of pyruvate kinase together with a sitmulation of gluconeogenesis. Insulin (10 nM) antagonized the effect of suboptimal doses of glucagon or cyclic AMP and of even maximal doses of epinephrine, on both pyruvate kinase activity and on gluconeogenesis. These observations can be explained by a phosphorylation of pyruvate kinase by cyclic-AMP-dependent protein kinase, as described by Ljungström et al. [(1974) Biochim. Biophys. Acta 358, 289-298] in a reconstructed system. They offer a molecular explanation for the hormonal control of gluconeogenesis. Glucose caused an inhibition of gluconeogenesis with no corresponding change in pyruvate kinase activity.

Animals↗
PubMed1985

Inhibition of cyclic AMP-dependent protein kinase-induced changes in the kinetic properties of hepatic pyruvate kinase by the specific cyclic AMP antagonist, the (Rp)-diastereomer of adenosine cyclic 3',5'-phosphorothioate.

The effects of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)- and (Rp)-cAMPS, on the kinetic properties of pyruvate kinase were studied in hepatocytes isolated from fed rats. Incubation of the cells with the cAMP-dependent protein kinase agonist, (Sp)-cAMPS, produced a concentration-dependent increase in S0.5 for phosphoenolpyruvate, but had no effect on Vmax. The (Sp)-cAMPS-treated enzyme was more sensitive to inhibition by alanine and ATP and, under the same conditions, was less responsive to activation by fructose-1,6-bisphosphate when assayed at a subsaturating phosphoenolpyruvate concentration. Incubation of the hepatocytes with only the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, produced no change in any kinetic parameters, but did suppress the (Sp)-cAMPS- or glucagon-induced increase in the S0.5 for phosphoenolpyruvate with IC50 values of 10 microM and 5 microM (Rp)-cAMPS. (Rp)-cAMPS is exerting an effect on the kinetic properties of pyruvate kinase through inhibition of cAMP-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1980

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.

Adenosine↗
PubMed1984

[Pyruvate kinase in the bovine adrenal cortex].

Pyruvate kinase from bovine adrenal cortex was purified to an electrophoretically homogeneous state. The molecular weight of the native enzyme is about 230 000, that of one subunit is 57 000. The maximal values of the pyruvate kinase initial reaction rate were obtained in 50 mM imidazole-acetate buffer within the pH range of 6.8 to 7.0. The curve of the initial pyruvate kinase reaction rate versus phosphoenolpyruvate (PEP) and ADP concentrations is hyperbolic and obeys the Michaelis-Menten kinetics with Km for PEP and ADP of 0.055 X 10(-3) M and 0.25 X 10(-3) M, respectively. The enzyme is activated by Mn2+ and Co2+ by 43 and 38%, respectively. IDP, GDP, and UDP may be used as analogs of ADP. The enzyme is not activated by fructose-1.6-diphosphate and is inhibited by L-phenylalanine and ATP.

Adenosine Triphosphate↗
PubMed1977

Affinity labeling of rabbit muscle pyruvate kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Rabbit muscle pyruvate kinase is irreversibly inactivated upon incubation with the adenine nucleotide analogue, 5'-p-fluorosulfonylbenzoyladenosine. A plot of the time dependence of the logarithm of the enzymatic activity at a given time divided by the initial enzymatic activity(logE/Eo) reveals a biphasic rate of inactivation, which is consistent with a rapid reaction to form partially active enzyme having 54% of the original activity, followed by a slower reaction to yield totally inert enzyme. In addition to the pyruvate kinase activity of the enzyme, modification with 5'-p-fluorosulfonylbenzoyladenosine also disrupts its ability to catalyze the decarboxylation of oxaloacetate and the ATP-dependent enolization of pyruvate. In correspondence with the time dependence of inactivation, the rate of incorporation of 5'-p-[14C]fluorosulfonylbenzoyladenosine is also biphasic. Two moles of reagent per mole of enzyme subunit are bound when the enzyme is completely inactive. The pseudo-first-order rate constant for the rapid rate is linearly dependent on reagent concentration, whereas the constant for the slow rate exhibits saturation kinetics, suggesting that the reagent binds reversibly to the second site prior to modification. The adenosine moiety is essential for the effectiveness of 5'-p-fluorosulfonylbenzoyladenosine, since p-fluorosulfonylbenzoic acid does not inactivate pyruvate kinase at a significant rate. Thus, the reaction of 5'-p-fluorosulfonylbenzoyladenosine with pyruvate kinase exhibits several of the characteristics of affinity labeling of the enzyme. Protection against inactivation by 5'-p-fluorosulfonylbenzoyladenosine is provided by the addition to the incubation mixture of phosphoenolpyruvate. Mg-ADP or Mg2+. In contrast, the addition of pyruvate, Mg-ATP, or ADP and ATP alone has no effect on the rate of inactivation. These observations are consistent with the postulate that the 5'-p-fluorosulfonylbenzoyladenosine specifically labels amino acid residues in the binding region of Mg2+ and the phosphoryl group of phosphoenolpyruvate which is transferred during the catalytic reaction. The rate of inactivation increases with increasing pH, and k1 depends on the unprotonated form of an amino acid residue with pK = 8.5. On the basis of the pH dependence of the reaction of pyruvate kinase with 5'-p-fluorosulfonylbenzoyladenosine and the elimination of cysteine residues as possible sites of reaction, it is postulated that lysyl or tyrosyl residues are the most probably candidates for the critical amino acids.

Adenosine↗
PubMed1981

L-phenylalanine induced changes of sulfhydryl reactivity in rabbit muscle pyruvate kinase.

Reactivity of sulfhydryl groups in rabbit muscle pyruvate kinase toward 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) was studied in the presence of activating divalent metal ions, substrate, substrate analogue, and the allosteric inhibitor, L-Phe. The pattern of sulfhydryl modification in various complexes of pyruvate kinase was consistent with the extent of enzyme inactivation by DTNB under very similar conditions. The sulfhydryl reactivity of Mg(II)-, Co(II)-, and Mn(II)-substituted pyruvate kinase toward DTNB depended upon the nature of the activating divalent metal ions used in the following order of increasing potency, Mg(II) less than Mn(II) less than Co(II), which is inversely related to the order of catalytic efficiency of these metal ions at alkaline pH. Similar optical spectra and the patterns of sulfhydryl modification by DTNB of the metal derivatives of pyruvate kinase were observed upon the binding of the substrate, phosphoenolpyruvate (PEP), or the substrate analogue, phosphoglycolate, which also provided a complete protection against enzyme inactivation by DTNB. L-PHe, on eht other hand, deprotected the enzyme from inactivation and further sulfhydryl modification by DTNB in the presence of PEP with the following order of potency depending upon the activating metal ions, Mn(II) less than Co(II) less than Mg(II), which parallels the order of metal dependency of L-Phe inhibition of this enzyme. L-Ala, which reverses the L-Phe inhibition of Mg(II)- or Co(II)-activated enzyme, restored the protective effect of PEP in the presence of L-Phe. The different patterns of sulfhydryl reactivity toward Mn(II)-enzyme (hyperbolic) and Mg(II)-enzyme (sigmoidal) correspond well with their kinetic patterns in the presence of L-Phe, indicating the presence of different conformational states between these two metal-enzyme complexes. These results led us to conclude that enzyme sulfhydryl reactivity toward DTNB can be used as a valid index for allosteric conformational changes of rabbit muscles pyruvate kinase.

Alanine↗
PubMed2001

Involvement of both phosphatidylinositol 3-kinase and p44/p42 mitogen-activated protein kinase pathways in the short-term regulation of pyruvate kinase L by insulin.

Pyruvate kinase L (PK-L) is a key regulatory enzyme of the hepatic glycolytic/gluconeogenic pathway that can be dephosphorylated and activated in response to insulin. However, the signaling cascades involved in this insulin effect have not been established. In this work we have investigated the potential involvement of phosphatidylinositol 3-kinase (PI 3-K) and p44/p42 mitogen-activated protein kinase (MAPK) pathways in the short-term modulation of PK-L by insulin in primary cultures of rat hepatocytes. Wortmannin, at a concentration of 100 nM, caused a marked inhibition of the PI 3-K/protein kinase B pathway, which became complete at 500 nM wortmannin. Likewise, wortmannin at 100 and 500 nM, elicited partial and total inhibitions of insulin-mediated activation of PK-L, respectively. However, this PI 3-K inhibitor also reduced insulin-mediated phosphorylation of p44/p42 MAPK in cultured rat hepatocytes, indicating that both the PI 3-K and MAPK pathways could be involved in PK-L activation by insulin. Three facts appear to reinforce this hypothesis: 1) the selective and complete inhibition of the PI 3-K/protein kinase B pathway by LY294002 (50 microM) was accompanied by a partial blockade of insulin-induced PK-L activation; 2) when signaling through the MAPK cascade was selectively suppressed by the presence of PD98059 (50 microM), a 50% reduction of insulin-induced activation of PK-L was observed; and 3) the effect of PD98059 (50 microM) on PK-L activation was reinforced by the additional presence of 100 nM wortmannin. We also observed that the blockade of p70 S6-kinase by rapamycin did not affect the activation of PK-L by insulin. From these findings it can be concluded that both PI 3-K and MAPK pathways, but not p70 S6-kinase, are involved in the short-term activation of PK-L by insulin in rat hepatocytes.

Androstadienes↗
PubMed1985

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, phosphoenolpyruvate 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 phosphoenolpyruvate 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.

Adenosine Diphosphate↗
PubMed1982

An immunological study of the interaction of ligands with pyruvate kinase of Neurospora crassa.

Antibodies against pyruvate kinase of Neurospora crassa, induced in rabbits, were used to monitor the interaction of ligands with this enzyme. The technique of microcomplement fixation was employed to probe for conformational alterations elicited by binding of substrates (phosphoenolpyruvate (PEP) and adenosine diphosphate), the allosteric activator (fructose 1,6-diphosphate), and the inhibitor (valine). On binding of PEP and valine to pyruvate kinase a pronounced reduction in the extent of complement fixation was observed. The second substrate, ADP, had no effect while FDP elicited a moderate suppression of complement fixation. These results suggest that as a consequence of conformational changes induced by PEP and valine, some antigenic determinants on the surface of pyruvate kinase are rendered inaccessible to the antibodies.

Adenosine Diphosphate↗
PubMed1992

Some kinetic properties of pyruvate kinase from Trypanosoma brucei.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1993

Purification and characterization of human meningioma M2-type pyruvate kinase.

The M2-type pyruvate kinase was purified from human meningioma by ammonium sulfate precipitation, followed by ion exchange and affinity chromatography. The specific activity of the purified enzyme was 33.4 U/mg with a yield of 6.5%. The enzyme gave a single band with 63,000 +/- 2000 Da upon SDS polyacrylamide gel electrophoresis. On cellulose acetate electrophoresis zymograms, the purified enzyme (M2) showed a single band, while crude extracts gave two broad bands corresponding to pyruvate kinase isozymes. The pI value of purified enzyme was found to be 6.9. With phosphoenol pyruvate as substrate the purified enzyme showed sigmoidal kinetics, while in the presence of 0.6 mM fructose 1,6-diphosphate as modulator it gave a hyperbolic saturation curve with a Km value of 0.53 mM.

Chromatography, Affinity↗
PubMed1987

Effects of chronic renal failure on the regulation of pyruvate kinase.

The effects of chronic renal failure on the enzyme activity of pyruvate kinase and the mRNA level of this enzyme were studied in 7 out of 8 nephrectomized rats. The mRNA level was measured by RNA-DNA dot blot hybridization, using cloned pyruvate kinase cDNA as hybridized probe. Neither the activity of M1-type pyruvate kinase nor the level of this enzyme in rat gastrocnemius muscle was affected by chronic renal failure, whereas L-type pyruvate kinase enzyme activity in uremic rat liver was lower than that in control at both fasted and refed states. The levels of L-type pyruvate kinase mRNA were not different between two groups at the fasted state. Induction of L-type pyruvate kinase mRNA after high carbohydrate diet refeeding was suppressed proportionally to the severity of chronic renal failure, which was expressed by the serum creatinine concentrations (r = -.876, P less than .005). These results indicate that the suppression of L-type pyruvate kinase activity in uremia was partly reflected by the decreased accumulation of this enzyme mRNA. There was a significantly negative correlation between L-type pyruvate kinase mRNA levels and plasma glucagon/insulin ratios (r = -.719, P less than .05). Hyperglucagonemia in uremia might play a major role in this suppression.

Animals↗
PubMed1992

Energetics of allosteric regulation in muscle pyruvate kinase.

The regulatory mechanism of rabbit muscle pyruvate kinase has been studied as a function of temperature in conjunction with phenylalanine, the allosteric inhibitor. The inhibitory effect of phenylalanine is modulated by temperature. At low temperatures, the presence of phenylalanine is almost inconsequential, but as the temperature increases so does the phenylalanine-dependent inhibition of the kinetic activity. In addition, the presence of phenylalanine induces cooperativity in the relation between velocity and substrate concentration. This effect is especially pronounced at elevated temperature. The kinetic data were analyzed using an equation that describes the steady-state kinetic velocity data as a function of five equilibrium constants and two rate constants. Van't Hoff analysis of the temperature dependence of the equilibrium constants determined by nonlinear curve fitting revealed that the interaction of pyruvate kinase with its substrate, phosphoenolpyruvate, is an enthalpy-driven process. This is consistent with an interaction that involves electrostatic forces, and indeed, phosphoenolpyruvate is a negatively charged substrate. In contrast, the interaction of pyruvate kinase with phenylalanine is strongly entropy driven. These results imply that the binding of phenylalanine involves hydrophobic interaction and are consistent with the basic concepts of strengthening of the hydrophobic effect with an increase in temperature. The effect of phenylalanine at high temperatures is the net consequence of weakening of substrate-enzyme interaction and significant strengthening of inhibitor binding to the inactive state of pyruvate kinase. The effects of salts were also studies. The results show that salts also exert a differential effect on the binding of substrate and inhibitor to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1983

Postnatal changes in canine erythrocyte pyruvate kinase isozymes.

The isozyme pattern of pyruvate kinase in canine erythrocytes changes following birth. These changes have been followed by electrophoretic, immunologic, and kinetic measurements of the isozymes. At birth, a mixture of isozymes is present consisting of the M2 isozyme and hybrid molecules containing M2 and R subunits. With increasing animal age, the content of M2 subunits decreases and the content of R subunits increases. At 6 months of age, the isozyme pattern is indistinguishable from that of adult erythrocytes which contain only the R tetramer. We conclude that there is a switch in erythrocyte pyruvate kinase gene expression during the first 6 months of postnatural life. The existence of hybrid molecules during the switch indicates that both M2 and R genes are expressed within each erythroid precursor cell. The developmental changes in erythrocyte pyruvate kinase are consistent with the role of this enzyme in the regulation of the oxygen-transport function of canine hemoglobin by 2,3-diphosphoglycerate in the postnatal period.

2,3-Diphosphoglycerate↗
PubMed1981

Single subunits of Sepharose-bound pyruvate kinase are inactive.

Bovine skeletal muscle pyruvate kinase was covalently coupled to Sepharose that had previously been activated by low concentrations of cyanogen bromide. Reaction conditions were chosen such that essentially all tetrameric enzyme molecules were covalently bound via a single subunit. Denaturation of the immobilized enzyme with guanidine hydrochloride followed by removal of noncovalently bound subunits amd denaturant resulted in essentially no enzymatic activity remaining bound to the resin. Thus, single immobilized subunits of bovine pyruvate kinase were inactive. Sepharose-bound enzymatic activity could be recovered by adding soluble renaturing enzyme subunits to the immobilized monomers. The former combine noncovalently with the latter, presumably resulting in re-formation of bound tetramers, and an average recovery of 61% of the original matrix-bound activity was observed. While interactions with other enzyme subunits appear to be necessary for catalytic activity of bovine muscle pyruvate kinase, these subunit interactions apparently can be provided by chemically modified subunits. Soluble, renaturing subunits from enzyme that had been inactivated by treatment with trinitrobenzenesulfonic acid were able to interact with matrix-bound single subunits, thereby restoring the enzymatic activity of the latter.

Animals↗
PubMed1976

Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid.

Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.

Alkylation↗