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PubMed1990

Pyruvate kinase from cytosolic fractions of the Ehrlich ascites tumour, normal mouse liver and skeletal muscle.

Comparative studies on cytosolic pyruvate kinase (PK, EC 2.7.1.40) from the Ehrlich ascites tumour, mouse liver and skeletal muscle, revealed the presence of two pyruvate kinase fractions: fraction A, salted out by ammonium sulphate between 21-30% saturation and predominant in the liver (type L); fraction B, salted out between 51-60% saturation and predominant in the tumour (type M2) or skeletal muscle (type M). The sigmoidal kinetics revealed in liver pyruvate kinase only were reconstructed in the mixture of both liver fractions A and B and characterized separately with linear kinetics in a double-reciprocal plot. L-Cysteine inhibited the neoplastic fraction B of pyruvate kinase only by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate. Stearic acid altered kinetic parameters of both fractions A and B of pyruvate kinase from the muscle and liver, but not from the tumour. This suggests that tumours contain a pyruvate kinase variant, characterized by a greater affinity to 2-phosphoenolpyruvate as the main substrate and by a different sensitivity to low-molecular effectors, in comparison with types L, M or M2 of pyruvate kinase from normal tissues.

Ammonium Sulfate↗
PubMed1988

ADP-ribosylation suppresses phosphorylation of the L-type pyruvate kinase.

L-type pyruvate kinase (EC 2.7.1.40) purified from pig liver was ADP-ribosylated by incubation with NAD and ADP-ribosyltransferase purified from hen liver nuclei. Maximal incorporation of the ADP-ribose moiety from NAD into the L-type pyruvate kinase was 0.98 mol/mol of subunit. The Km values for NAD and L-type pyruvate kinase were 0.17 mM and 9.7 microM, respectively. ADP-ribosylation of the L-type pyruvate kinase resulted in suppression of the subsequent phosphorylation catalyzed by cAMP-dependent protein kinase. The ADP-ribosylation-induced suppression of phosphorylation of the L-type pyruvate kinase also resulted in suppression of the phosphorylation-induced inactivation. Amino acid analysis, after exhaustive sequential digestion of ADP-ribosyl-L-type pyruvate kinase with pepsin, aminopeptidase M and carboxy-peptidase B showed arginine to be the ADP-ribose-accepting amino acid. These results together with finding of the ADP-ribosyltransferase activity in mammalian liver cytosol (Moss, J. and Stanley, S.J. (1981) J. Biol. Chem. 256, 7830-7833) suggest that ADP-ribosylation may participate in the regulation of the L-type pyruvate kinase activity through changes in the rate of phosphorylation.

Adenosine Diphosphate Ribose↗
PubMed1993

Pyruvate kinase from Lactobacillus bulgaricus: possible regulation by competition between strong and weak effectors.

The pyruvate kinase from Lactobacillus bulgaricus has been purified to homogeneity. The native enzyme is composed of four probably identical subunits of relative molecular mass M(r) 72,000 +/- 4,000. The unique N-terminal amino acid sequence is homologous to those of other pyruvate kinases, especially of type I and II enzymes from Escherichia coli. The saturation of the pyruvate kinase from Lactobacillus bulgaricus is hyperbolic for ADP and cooperative for the other substrate phospho-enol-pyruvate. The enzyme is strongly activated by glucose-6-phosphate, ribose-5-phosphate, and fructose-6-phosphate, which increase the affinity for phospho-enol-pyruvate. These activators seem to stabilize the same state of the enzyme, since their maximum activations are not additive, but their partial activations can be cumulated. Pyruvate kinase is also weakly activated by AMP and inhibited by fructose-1,6-bisphosphate. However, both AMP and fructose-1,6-bisphosphate act as strong inhibitors in the presence of a strong activator, because these weak effectors suppress the activation by glucose-6-phosphate, ribose-5-phosphate, or fructose-6-phosphate. This mutual exclusion of strong and weak effectors, which appears as an original regulatory mechanism, could reflect either the binding of different effectors to different interacting sites or their competition for a unique polyvalent regulatory site in the pyruvate kinase from Lactobacillus bulgaricus.

Adenosine Diphosphate↗
PubMed1978

Alterations in the pyruvate dehydrogenase complex during adaptation to glucose by Neurospora.

A 20-fold induction of the pyruvate dehydrogenase complex, pyruvate dehydrogenase (EC 1.2.4.1) plus dihydrolipoate S-acetyltransferase, (lipoyltransacetylase) (EC 2.3.1.12) plus dihydrolipoyl dehydrogenase, NADH : lipoamide oxidoreductase, (EC 1.6.4.3), from a specific activity of 3.5-65.0 was observed in mitochondrial extracts during adaptation of Neurospora to glucose from acetate media. The extent of ATP-dependent, time-dependent inactivation of the pyruvate dehydrogenase complex was approximately the same in both acetate- and glucose-grown cells, thereby indicating that the low pyruvate dehydrogenase complex activities in acetate-grown cells did not represent phosphorylated pyruvate dehydrogenase complex molecules. High levels of dihydrolipoyl transacetylase (EC 2.3.1.12) were observed in mitochondrial extracts from acetate-grown cells; this lipoyltransacetylase was analyzed on sucrose density gradients and found to be associated with the pyruvate dehydrogenase complex. Digitonin fractionation of mitochondria revealed that both the pyruvate dehydrogenase complex and lipoyltransacetylase were primarily associated with the mitochondrial outer membrane.

Acetates↗
PubMed1994

Zonation of gluconeogenesis from lactate and pyruvate in the rat liver studied by means of anterograde and retrograde bivascular perfusion.

Gluconeogenesis from lactate and pyruvate and associated parameters were investigated in the bivascularly and hemoglobin-free perfused rat liver. The substrates were infused either via the portal vein (anterograde perfusion mode), via the hepatic vein (retrograde mode) or via the hepatic artery (anterograde and retrograde modes). The rates of lactate and pyruvate infusion were 10.3 and 3.5 mumol min-1 g-1, respectively. The metabolic rates measured when the substrates were infused into the hepatic artery were referred to the cellular spaces accessible in each perfusion mode. The following results were obtained when the substrates were infused into the hepatic artery: (1) gluconeogenesis from lactate was equal to 2.08 +/- 0.2 mumol min-1 ml-1 in the retrograde mode and 1.33 +/- 0.08 mumol min-1 ml-1 in the anterograde mode (P = 0.019); (2) gluconeogenesis from pyruvate was equal to 0.66 +/- 0.11 mumol min-1 ml-1 in the retrograde mode and 0.7 +/- 0.11 mumol min-1 ml-1 in the anterograde mode (P = 0.78); (3) oxygen uptake increase with lactate was 1.75 +/- 0.14 mumol min-1 ml-1 in the retrograde mode and 1.05 +/- 0.07 mumol min-1 ml-1 in the anterograde mode (P = 0.002); (4) oxygen uptake increase with pyruvate was equal to 0.59 mumol min-1 ml-1 in the retrograde mode and 0.57 +/- 0.05 mumol min-1 ml-1 in the anterograde mode (P = 0.73); (5) pyruvate production from lactate was 0.28 +/- 0.06 mumol min-1 ml-1 in the retrograde mode and 0.39 +/- 0.05 mumol min-1 ml-1 in the anterograde mode (P = 0.28); (6) lactate production from pyruvate was equal to 0.52 +/- 0.05 mumol min-1 ml-1 in the retrograde mode and 0.99 +/- 0.08 mumol min-1 ml-1 in the anterograde mode (P < 0.001). Since only periportal cells are supplied with substrates when they are infused via the hepatic artery in retrograde perfusion, these results allow the conclusion that gluconeogenesis from lactate predominates in periportal hepatocytes. When pyruvate is the sole substrate, however, gluconeogenesis in periportal and perivenous cells presents no difference.

Animals↗
PubMed1979

Regulation of glucose formation from lactate and pyruvate in isolated tubules of chicken kidney.

1. Isolated kidney tubules from chicken have been used to study the actions of ethanol, ouabain and aminooxyacetate on glucose formation from lactate and pyruvate. 2. In kidney tubules from well-fed chickens the rate of glucose production from lactate was higher than from pyruvate. Ethanol (10 mM) and ouabain (0.1 mM) were found to increase glucose formation from pyruvate but not from lactate. 3. It is concluded that in the presence of ethanol the fluxes of pyruvate through pyruvate dehydrogenase are in favour of the pyruvate carboxylase reaction restricted. 4. Glucose formation from lactate is decreased by aminooxyacetate (0.1 mM) and ouabain (0.1 mM). 5. Aminooxyacetate inhibited glucose formation from lactate, although chicken phosphoenolpyruvate carboxykinase is located intramitochondrially. 6. The results indicate that the effect of aminooxyacetate like that of ouabain is caused by the restricted formation of pyruvate.

Aminooxyacetic Acid↗
PubMed2004

Exogenous pyruvate prevents stress-evoked suppression of mitogen-stimulated proliferation.

Although the phenomenon that psychological stress influences disease onset and progression is well established, the mechanisms underlying stress-evoked compromise of immune function remain unspecified. To test the hypothesis that energetic shortages compromise immunity, we evaluated the effectiveness of pyruvate, a metabolic supplement, to prevent stress-evoked suppression of mitogen-stimulated splenocyte proliferation. Male C57BL/6 mice were subjected to 2h of restraint once daily for 14 days. Consistent with previous studies, mitogen-stimulated splenocyte proliferation was reduced after restraint; in contrast, mice that received pyruvate injections immediately following each episode of restraint did not reduce splenocyte proliferation. In addition, restraint-evoked corticosterone elevation did not habituate in animals treated with pyruvate, suggesting that glucocorticoids are not exclusively immunosuppressive. The ratio of pyruvate to lactate, an index of aerobic metabolism, was elevated in mice exposed to restraint suggesting that mice exposed to restraint were preferentially using aerobic metabolism and producing more ATP per unit of pyruvate than non-restrained mice. Furthermore, two of the effective doses of pyruvate (0.5 and 500.0mg/kg) altered glucose levels suggesting a metabolic function of the supplement. Although several different mechanisms could possibly mediate the changes in splenocyte proliferation, these results support the hypothesis that stress-evoked immunosuppression may be a function of metabolic energy shortages and can be prevented via pyruvate supplementation.

Analysis of Variance↗
PubMed2006

Enhancement of pyruvate productivity in Torulopsis glabrata: Increase of NAD+ availability.

This study aimed at increasing the pyruvate productivity from a multi-vitamin auxotrophic yeast Torulopsis glabrata, by increasing the availability of NAD+. We examined two strategies for increasing availability of NAD+. To supplement nicotinic acid (NA), the precursor of NAD+; and to increase the activity of alcohol dehydrogenase integrating with addition acetaldehyde as exterior electron acceptor. The addition of 8 mg l(-1) NA to the fermentation medium resulted in a significant increase in the glucose consumption rate (48.4%) and the pyruvate concentration (29%). An ethanol-utilizing mutant WSH-13 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the alcohol dehydrogenase activity of the mutant WSH-13 increased about 110% and the mutant could utilize ethanol as the sole carbon source for growth (1.8 g l(-1) dry cell weight). When growing with glucose, the addition of 4 mg l(-1) acetaldehyde to the mutant WSH-13 culture broth led to a significant increase in the glucose consumption rate (26.3%) and pyruvate production (22.5%), but the ratio of NADH/NAD+ decreased to 0.22. Acetaldehyde did not affect the glucose and energy metabolism at high dissolved oxygen (DO) concentration. However, at lower DO concentration (20%), maintaining the acetaldehyde concentration in the mutant culture broth at 4 mg l(-1) caused an increased NAD+ concentration but a decreased NADH concentration. As a consequence, the pyruvate production rate, the pyruvate yield on glucose and the pyruvate concentration were 68, 44 and 45% higher, respectively, than the corresponding values of the control (without acetaldehyde). The strategy for increasing the glycolytic flux and the pyruvate productivity in T. glabrata by increasing the availability of NAD+ may provide an alternative approach to enhance the metabolites productivity in yeast.

Biological Availability↗
PubMed2006

Pyruvate provides cardioprotection in the experimental model of myocardial ischemic reperfusion injury.

The present study was designed to evaluate the cardioprotective potential of pyruvate and to characterize the mechanism underlying the protection. Wistar albino rats were randomly divided into three groups. Two groups were administered saline orally (sham, ischemia-reperfusion (I-R) control group) and animals of third group received pyruvate (500 mg/kg) for 4 weeks. On the 29th day, animals of the I-R control and pyruvate treated groups underwent 45 min of occlusion of the left anterior descending (LAD) coronary artery and were thereafter reperfused for 60 min. In the I-R control group, a significant cardiac necrosis, depressed mean arterial pressure (MAP) and heart rate (HR), decline in myocardial antioxidant status and elevation in lipid peroxidation were observed as compared to sham control. Pyruvate treatment restored the myocardial antioxidant status and favorably modulated the altered MAP as compared to I-R control. Furthermore, I/R-induced lipid peroxidation was significantly inhibited by pyruvate treatment. These beneficial cardioprotective effects translated into significant improvement in MAP. Histopathological examination and restored specific myocardial injury marker CK-MB isoenzyme activity further confirmed protective effects of pyruvate. In conclusion, our study has demonstrated that the beneficial effect of pyruvate likely results from improved MAP and suppression of oxidative stress.

Animals↗
PubMed2003

Glutamate-pyruvate transaminase protects against glutamate toxicity in hippocampal slices.

Elimination of glutamate through enzymatic degradation is an alternative to glutamate receptor blockade in preventing excitotoxic neuronal injury. Glutamate pyruvate transaminase (GPT) is a highly active glutamate degrading enzyme that requires pyruvate as a co-substrate. This study examined the ability of GPT to protect neurons of the hippocampal slice preparation against glutamate toxicity. Two methods were used to elevate the concentration of glutamate in the peri-neuronal space. In an endogenous release paradigm, slices were incubated with 100-500 microM L-trans-pyrrolidine-2,4-dicarboxylate (PDC), an inhibitor of glutamate re-uptake. One hour of exposure to PDC in normal, pyruvate-free slice maintenance medium caused a dose dependent increase in neuronal death assessed 24 h later by propidium iodide uptake in dead cell nuclei. GPT (10 U/ml) decreased neuronal death caused by exposure to PDC at all PDC concentrations tested. Neuroprotection in this model was not dependent on added or non-physiologic levels of pyruvate. In a different paradigm, glutamate was added directly to the normal, pyruvate-free slice maintenance medium and not rinsed away, exposing the slices to a range of 1-5 mM glutamate for an extended period. Twenty-four hours later, neuronal death was again assessed by propidium iodide uptake. GPT was again neuroprotective, decreasing neuronal death in the range from 3 to 5 mM glutamate. In the setting of incubation with this large load of glutamate, neuroprotection by GPT was enhanced by adding pyruvate to the medium. GPT is an effective neuroprotectant against glutamate excitotoxicity. When exposure is limited to endogenously released glutamate, neuroprotection by GPT is not dependent on added pyruvate.

Alanine Transaminase↗
PubMed2003

Neuroprotective effects of pyruvate in the quinolinic acid rat model of Huntington's disease.

The neuroprotective effects of pyruvate, the end metabolite of glycolysis, were studied in an animal model of Huntington's disease (HD). Intrastriatal injection of quinolinic acid (QA) caused widespread damage to rat striatum as determined from cresyl violet staining and immunohistochemical analysis. Intraperitoneal administration of pyruvate at doses of 500-1000 mg/kg significantly reduced striatal lesions induced by QA. A lower pyruvate concentration of 250 mg/kg was not protective; however, quadruple applications at this dosage was effective in reducing lesion volumes. The protective effects of pyruvate were found over a range of times, from application at the time of QA injection to 1 h post-administration; however, no protection was conferred if pyruvate was applied 30 min prior to QA injection or 3 h post-administration. We also found pyruvate protects different types of striatal neurons against QA toxicity including GABAergic projection neurons, cholinergic interneurons and NADPH-diaphorase interneurons. These results suggest that pyruvate may be effective in reducing neuronal damage in HD.

Animals↗
PubMed1998

Mechanism of preservation of myocardial calcium channel function by pyruvate cardioplegic solution.

We evaluated the effects of adding pyruvate to a cardioplegic solution on the preservation of the dihydropyridine-sensitive calcium (Ca2+) current responses to beta-adrenergic stimulation in rabbit cardiac myocytes by measurement of single-channel open probability. Single ventricular myocytes were isolated and stored in St. Thomas' solution with or without pyruvate at 4 degrees C for 2, 6, 12, or 24 hours, and cell-attached single Ca2+ channel currents recordings were made at 20 degrees to 22 degrees C after each storage period. When 0.1 micromol/L isoproterenol (ISO) was applied to the cells, the percent mean open probability of the Ca2+ channels tested in freshly isolated cells was 181% +/- 27% (n = 12) of control values. These responses decreased with an increasing duration of the hypothermic storage and were only 112% +/- 22% (n = 5) of control values after 24 hours of storage in the absence of pyruvate. Conversely, the responses were significantly preserved, to as much as 143% +/- 17% (n = 7), in the presence of 10 mmol/L pyruvate in the storage solution. The application of forskolin to stimulate adenylate cyclase or a membrane-permeable cyclic adenosine monophosphate mimicked the effects of ISO when the myocytes were stored with pyruvate. Pyruvate did not alter the open-channel kinetics or single-channel conductance and lacked any apparent direct effect on the Ca2+ channel activity. We suggest that pyruvate added to the hypothermic storage solution preserves the high-energy phosphates in myocytes that are responsible for Ca2+ channel phosphorylation via beta-adrenergic stimulation. (J Lab

Animals↗
PubMed1986

Pyruvate attenuation of hypoxia damage in isolated working guinea-pig heart.

Function and various parameters of myocardial substrate and energy metabolism were measured in preload-controlled isolated working guinea-pig hearts perfused with normoxic (95% O2) and hypoxic (30 to 45% O2) Krebs-Henseleit buffers ([Ca2+] = 1.25 mM). Energy-yielding substrates were glucose, pyruvate, lactate, and fatty acids (acetate, octanoate). Hypoxia typically produced an increase in coronary flow but a fall in cardiac oxygen uptake (MVO2); left ventricular pressure and work parameters as well as myocardial high energy phosphate levels were decreased while the releases of adenosine plus inosine (V (Ado + Ino)) and lactate were increased. Extra pyruvate (1 to 5 mM) as compared to physiologic concentrations of pyruvate (0.2 mM) produced a relative stabilization of left ventricular pressure and work parameters combined with an attenuation of V (Ado + Ino) provided 5 to 10 mM glucose was the cosubstrate. Coinfusion of 2-deoxyglucose, a nondegradable hexose, in presence of excess pyruvate as sole substrate was without effects on residual ventricular pump function. When 1 mM lactate plus 5 mM glucose were the substrates, hypoxic heart function was also depressed, V (Ado + Ino) was relatively increased, and post-hypoxic recovery of pressure parameters was impaired. Similarly, the fatty acid substrates tested seemed to adversely affect cardiac performance during hypoxia. Extra pyruvate in presence of glucose induced a fall in hypoxic myocardial lactate and alpha-glycerophosphate contents while cellular citrate reached millimolar levels. Obviously, utilizable amounts of glucose were required for pyruvate stabilization of the high flow hypoxic heart. The beneficial effects of pyruvate appeared to depend on a functioning glycolysis; other effects seemed to include redox-related changes in energy state and/or purine nucleoside metabolism as well as a possible citrate buffering of intracellular Ca2+ load.

Animals↗
PubMed2003

Mechanisms of pyruvate inhibition of oxidant-induced apoptosis in human endothelial cells.

We have recently demonstrated that the redox reactant pyruvate prevents hydrogen peroxide (H2O2)-induced endothelial apoptosis and that its anti-apoptotic feature is mediated partially through the mitochondrial compartment. However, little is known about molecular signal pathways that mediate the anti-apoptotic feature of pyruvate. A biochemical approach to elucidate such signal pathways was attempted in human umbilical vein endothelial cells (HUVECs). Effects of antioxidant pyruvate were compared with those of cytosolic reductant L-lactate, redox-neutral acetate, and malate-aspartate shuttle blocker aminooxyacetate. Various indices of endothelial apoptosis were correlated with cell viability. Submillimolar H2O2 caused >50% cell killing, as manifested by its oxidant insult. The massive cell death induced by H2O2 was inhibited by pyruvate but not by L-lactate or aminooxyacetate, suggesting a role of cytosolic NADH reducing equivalents, possibly via stimulated oxidant generation. The induction and nuclear translocation of p53 by H2O2 was blocked by pyruvate and appeared to be somewhat enhanced by L-lactate or aminooxyacetate in association with oxidant generation. Nuclear translocation of p53 accompanied the transactivation of bax and downregulation of bcl-2. The pyruvate-related redox manipulation inhibited the H2O2-induced p53 activation, restored the downregulated bcl-2 and the upregulated bax, and hence enhanced the bcl-2/bax expression ratio. In contrast, L-lactate, acetate, or aminooxyacetate had no such effect. These results indicate that pyruvate could modulate key regulatory signal pathways in cytosol and mitochondrial matrix, thereby inactivating endothelial death pathways. Furthermore, it is suggested that stabilizing the expression of bcl-2 and bax genes by metabolic antioxidants may be an effective strategy for endothelial protection against oxidative stress.

Acetates↗
PubMed1998

Adrenocorticotropic hormone therapy for infantile spasms alters pyruvate metabolism in the central nervous system.

To clarify the mechanism of action of adrenocorticotropic hormone (ACTH) in treating infantile spasms, we evaluated the effects of ACTH on the metabolism of pyruvate in the central nervous system (CNS) of children with infantile spasms. We measured the levels of lactate and pyruvate in cerebrospinal fluid (CSF) and serum, before and during ACTH treatment in 12 children with infantile spasms. We evaluated statistically any correlation between the observed metabolic changes and the clinical response of ACTH. ACTH therapy significantly elevated the levels of lactate and pyruvate in the CSF and serum. The effect was not dose-dependent. During ACTH therapy, the serum levels of lactate and pyruvate and the ratio of lactate to pyruvate (L:P ratio) were unrelated to these levels in CSF. Patients who showed a good initial response to treatment had a significantly higher CSF level of pyruvate and a lower L:P ratio during therapy than did those with a poor initial response. This is the first report that ACTH therapy administered for infantile spasms alters pyruvate metabolism in the CNS. This metabolic change may be involved in part in the action of ACTH in relieving infantile spasms.

Adrenocorticotropic Hormone↗
PubMed1993

Water-induced transitions in the K+ requirements for the activity of pyruvate kinase entrapped in reverse micelles.

The activity of pyruvate kinase was studied in reverse micelles formed with cetyltrimethylammonium bromide, n-octane, hexanol, and various amounts of water. In systems with 100% water, K+ is an essential activator of pyruvate kinase [Kachmar, J. F., & Boyer, P. D. (1953) J. Biol. Chem. 200, 669-683]; i.e., without and with K+, the activities observed were 0.07 and 300 mumol/(min.mg), respectively. In the micellar system with 3.6% water (v/v), pyruvate kinase exhibited an activity of about 45 mumol/(min.mg), in the absence of K+. The kcat was about 450 times larger than that in 100% water without K+. Km values for ADP and phosphoenolpyruvate differed, but not markedly from those in 100% water with or without K+. The kinetics of pyruvate kinase in reverse micelles were not affected by K+. The activity curve of pyruvate kinase in reverse micelles without K+ in a pH range of 6.0-8.5 was almost superimposable to that of the enzyme in 100% water with K+, and it differed drastically from that in 100% water without K+. The fluorescence emission spectra of pyruvate kinase in 100% water exhibited a blue shift of 3 nm upon the addition of ligands (Mg2+, phosphoenolpyruvate, and K+) that cause a transition of the enzyme to its active state. Without ligands, the entrapment of pyruvate kinase in reverse micelles with 3.0% water produced a blue shift of nearly 2 nm with respect to that of the enzyme in 100% water without ligands. As water was raised to 7.0% (v/v), the maximal emission shifted to longer wavelengths; these changes paralleled the appearance of the K(+)-dependent activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart.

The highly purfied pyruvate dehydrogenase complex (EC 1.2.4.1) and uncomplexed pyruvate dehydrogenase from bovine kidney and heart mitochondria were phosphorylated and inactivated with pyruvate dehydrogenase kinase and [gamma-32P]ATP. Tryptic digestion of the phosphorylated pyruvate dehydrogenase yielded three phosphopeptides, a mono- (site 1) and a di- (sites 1 and 2) phosphorylated tetradecapeptide and a monophosphorylated nonapeptide (site 3). The amino acid sequences of the three phosphopeptides were established to be Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser-Tyr-Arg, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asn-Pro-Gly-Val-Ser(P)-Tyr-Arg, and Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg. Phosphorylation proceeded markedly faster at site 1 than at sites 2 and 3, and phosphorylation at site 1 correlated closely with inactivation of pyruvate dehydrogenase. Complete inactivation of pyruvate dehydrogenase was associated with incorporation at site 1 of 1.0--1.6 mol of phosphoryl groups per mol of enzyme. Since pyruvate dehydrogenase is a tetramer (alpha2beta2) and since phosphorylation occurs only on the alpha subunit, the possibility of half-site reactivity is considered.

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↗