A novel reaction of S-adenosyl-L-methionine correlated with the activation of pyruvate formate-lyase

Conversion of the inactive form of pyruvate formate-lyase to the catalytically active enzyme is accomplished by the Fe-dependent ‘enzyme II’; reduced flavodoxin, S-adenosyl-L-methionine and the effector pyruvate are required. It was found that adenosylmethionine is reductively processed during activation of pyruvate formate-lyase to yield methionine, adenine and 5-deoxyribose. We suggest that transient adenosylation of enzyme II is required for its function as a converter enzyme.     

Pyruvate degradation via pyruvate formate-lyase (EC 2.3.1.54) and the enzymes of formate fermentation in the green alga Chlorogonium elongatum

Formate was formed in extracts of Chlorogonium elongatum via direct cleavage of pyruvate by a pyruvate formate-lyase (PFL, EC 2.3.1.54). The conversion of PFL to the catalytically active form required S-adenosylmethionine, ferric (2+), photoreduced deazariboflavin as reductant, pyruvate as allosteric effector and strict anaerobic conditions. At the optimum pH (pH 8.0), PFL catalyzed formate formation, pyruvate synthesis and the isotope exchange from [¹⁴C]formate into pyruvate with rates of 30.0, 1.5 and 1.2 nmol min^-1 mg^-1 protein, respectively. Treatment of the active enzyme with O₂ irreversibly inactivated PFL activity (half-time 2 min). In addition to PFL, the activities of phosphotransacetylase (EC 2.3.1.8), acetate kinase (EC 2.7.2.1), aldehyde dehydrogenase (CoA acetylating, EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1) were also detected in extracts of C. elongatum. The occurrence of these enzymes indicates pyruvate degradation via a formate-fermentation pathway during anaerobiosis of algal cells in the dark.Abbreviations DTT dithiothreitol - Hepes 4-(2-hydroxyethyl)-1-piperazine+ethane sulfonic acid - PFL pyruvate formate-lyase     

Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine.

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and G?nzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).     

The decarboxylation of S-adenosylmethionine by detergent-treated extracts of rat liver

1. The production of (14)CO(2) from S-adenosyl[carboxyl-(14)C]methionine by rat liver extracts was investigated. It was found that, in addition to the well-known cytosolic putrescine-activated S-adenosylmethionine decarboxylase, an activity carrying out the production of (14)CO(2) could be extracted from a latent, particulate or membrane-bound form by treatment with buffer containing 1% (v/v) Triton X-100 [confirming the report of Sturman (1976) Biochim. Biophys. Acta428, 56-69]. 2. The formation of (14)CO(2) by such detergent-solubilized extracts differed from that by cytosolic S-adenosylmethionine decarboxylase in a number of ways. The reaction by the solubilized extracts did not require putrescine and was not directly proportional to time of incubation or the amount of protein added. Instead, activity a showed a distinct lag period and was much greater when high concentrations of the extracts were used. The cytosolic S-adenosylmethionine decarboxylase was activated by putrescine, showed strict proportionality to protein added and the reaction proceeded at a constant rate. Cytosolic activity was not inhibited by homoserine or by S-adenosylhomocysteine, whereas the Triton-solubilized activity was strongly inhibited. 3. By using an acetone precipitate of Triton-treated homogenates as a source of the activity, it was found that decarboxylated S-adenosylmethionine was not present among the products of the reaction, although 5'-methylthioadenosine and 5-methylthioribose were found. Such extracts were able to produce (14)CO(2) when incubated with [U-(14)C]-homoserine, and (14)CO(2) production was greater when S-adenosyl[carboxyl-(14)C]methionine that had been degraded by heating at pH6 at 100 degrees C for 30min (a procedure known to produce mainly 5'-methylthioadenosine and homoserine lactone) was used as a substrate than when S-adenosyl[carboxyl-(14)C]methionine was used. 4. These results indicate that the Triton-solubilized activity is not a real S-adenosylmethionine decarboxylase, but that (14)CO(2) is produced via a series of reactions involving degradation of the S-adenosyl-[carboxyl-(14)C]methionine. It is probable that this degradation can occur via several pathways. Our results would suggest that part of the reaction occurs via the production of S-adenosylhomocysteine, which can then be converted into 2-oxobutyrate via the transsulphuration pathway, and that part occurs via the production of homoserine by an enzyme converting S-adenosylmethionine into 5'-methylthioadenosine and homoserine lactone.     

Anaerobic regulation of pyruvate formate-lyase from Escherichia coli K-12.

The anaerobic regulation of the gene encoding pyruvate formate-lyase from Escherichia coli was investigated. Expression of a pfl'-'lacZ protein fusion demonstrated that the gene is subject to a 12-fold anaerobic induction which can be stimulated a further 2-fold by the addition of pyruvate to the growth medium. Construction of a strain deleted for pfl verified that either pyruvate or a metabolite of glycolysis functions as an inducer of pfl gene expression. Complete anaerobic induction required the presence of a functional fnr gene product. However, the dependence was not absolute since a two- to threefold anaerobic induction could still be observed in an fnr mutant. These results could be confirmed immunologically by analyzing the levels of pyruvate formate-lyase protein present in cells grown under various conditions. It was also shown that pfl'-'lacZ expression was partially repressed by nitrate and that this repression was mediated by the narL gene product.     

S-adenosylmethionine-dependent enzyme activation

S-Adenosylmethionine (SAM)-dependent activations of pyruvate formate-lyase, lysine 2,3-aminomutase and cobalamin-dependent methionine synthase are discussed. In each case, cleavage of SAM is accompanied by the formation of a catalytically active enzyme. The chemistry of activation of these three enzymes falls into three distinct classes: generation of an essential enzyme radical (pyruvate formate-lyase), formation of a catalytically active 5'-deoxyadenosyl radical (lysine 2,3-aminomutase) and reductive methylation to form a required methylcobalamin complex (methionine synthase).     

Fermentative metabolism of pyruvate by Rhodospirillum rubrum after anaerobic growth in darkness.

Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.     

Dynamics of pyruvate metabolism in Lactococcus lactis.

The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid-product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis in continuous cultures subjected to step changes in dilution rate. Both shift-up and shift-down experiments were carried out, and these experiments showed that the enzyme pyruvate formate-lyase (PFL) plays a key role in the regulation of the shift. Pyruvate formate-lyase in vivo activity was regulated both at the level of gene expression and by allosteric modulation of the enzyme. A simple mathematical model was proposed to estimate the relative significance of the regulatory mechanisms involved.     

Phospholipids and the regulation of pyruvate dehydrogenase from rat adipocyte mitochondria

Aqueous dispersions of 4 out of 9 phospholipids added individually to the mitochondrial fraction from rat adipocytes altered the activity of pyruvate dehydrogenase in a dose-dependent manner from 1 to 300 microM. Phosphatidylserine increased and phosphatidylcholine, phosphatidylinositol and phosphatidylinositol-4-phosphate decreased enzyme activity. The stimulation of pyruvate dehydrogenase induced by phosphatidylserine may be reversed to below basal activity by phosphatidylinositol-4-phosphate and to basal activity by NaF, a pyruvate dehydrogenase phosphatase inhibitor. The inhibition of pyruvate dehydrogenase induced by phosphatidylinositol-4-phosphate may be restored to basal levels by the addition of calcium. These results suggest that phosphatidylserine activates pyruvate dehydrogenase activity through activation of the phosphatase, perhaps forming a phosphatidylserine-calcium complex. The inhibition by phosphatidylinositol-4-phosphate may be mediated by disruption of the enzyme complex. The phospholipids may play a physiological role in the regulation of pyruvate dehydrogenase activity.     

Roles of nifF and nifJ gene products in electron transport to nitrogenase in Klebsiella pneumoniae.

Crude extracts of the wild-type Klebsiella pneumoniae reduced C2H2 with either pyruvate or formate as reductant (specific activity, 3 nmol min-1 mg of protein-1), whereas crude extracts of nifF mutant were almost inactive (specific activity, 0.05). However, activity in the latter extracts was stimulated by adding Azotobacter chroococcum flavodoxin (specific activity, 10). Thus, nifF mutants may lack an electron transport factor. Crude extracts of nifJ mutants had about 20% of the wild-type level of active MoFe protein, and thus nifJ has a presumptive role in maintaining active MoFe protein. Studies on pyruvate or formate as reductants for nitrogenase in extracts of the nifJ mutants suggest in addition a role in electron input to nitrogenase for the following reasons. (i) Nitrogenase activity with these reductants was very low (specific activity, 0.06) and was not stimulated by extra MoFe protein or the flavodoxin. (ii) Activity was increased by adding a crude extract of a mutant lacking the structural nif genes (specific activity, 1) or a crude extract of the nifF mutant (specific activity, 4).     

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a Km for methionine of about 3-12 microM. In selected lines we have demonstrated a requirement for Mg2+ in addition to that needed to form the Mg X ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.     

The regulation of rat brain pyruvate kinase by estradiol-17 beta.

The effect of estradiol-17 beta on the activity of pyruvate kinase from rat brain was investigated at initial stages of hormone administration. After estradiol treatment the rise of pyruvate kinase activity was found in the firmly bound synaptosomal fraction, whereas pyruvate kinase activity in soluble and weakly bound fractions has been reduced. The activation of the enzyme was also discovered after glutaraldehyde fixation on synaptosomal membranes. The possible mechanism of extragenomic estradiol action is discussed.     

The viral protein sigma 3 participates in translation of late viral mRNA in reovirus-infected L cells.

Reovirus late (uncapped) mRNA was previously shown to be efficiently translated in vitro extracts prepared from infected cells but not from uninfected cells. We demonstrated that different fractions from infected cells can stimulate translation of late viral mRNA when added to uninfected extracts. The activity of the different fractions correlated with their relative content of the sigma 3 capsid protein; the fraction prepared by high-salt wash of the ribosomes had the highest specific activity. The activity present in this fraction was abolished by preincubation with an anti-sigma 3 serum. Purified sigma 3 protein also stimulated the translation of late viral mRNA, confirming that it was the factor involved. Altogether, these results suggest that this protein plays the role of a late-viral-mRNA-specific initiation factor. The absence of an inhibitory effect of sigma 3 on the translation of other mRNAs indicates that this protein is not directly involved in the inhibition of host and early viral mRNA translation that occurs in infected cells but that a second mechanism is probably operative.     

Biosynthesis of ethylene: Formation of ethylene from methional by a cell-free enzyme system from cauliflower florets

1. The formation of ethylene from cauliflower florets is stimulated by the addition of either methionine or its hydroxy analogue. 2. Formation of ethylene from these compounds may also be demonstrated in cell-free extracts, but the most rapid formation is achieved by the addition of methional. 3. Fractionation of such extracts has shown that both particulate and non-particulate fractions are necessary for the formation of ethylene from methionine or its hydroxy analogues, but only the non-particulate fraction is necessary for its formation from methional. 4. A study of this system has shown that the conversion of methional into ethylene requires the presence of two enzyme systems, the first generating peroxide and the second catalysing the conversion of methional into ethylene in the presence of peroxide. 5. The presence of a heat-stable factor in cauliflower extracts that is necessary for the full activity of the enzyme converting methional into ethylene has also been shown. 6. The nature of this factor is at the present unknown; it is not a metal nor is it identifiable with many of the known coenzymes.     

Some properties of pyruvate kinase extracted from Lycopersicon esculentum

Pyruvate kinase was extracted from Me₂CO-dried tissue of various parts of tomato plants. Recovery of the enzyme was improved by the inclusion of thiols in the extraction medium, and its stability was increased considerably in the presence of glycerol and to a lesser extent tetramethylammonium chloride. A phosphatase was present in the tissue extracts which hydrolyses phosphoenolpyruvate in the absence of added ADP. ATP inhibited pyruvate kinase but stimulated the phosphatase, while Mg²⁺ stimulated both enzymes. Data obtained suggest that tomato leaf pyruvate kinase has an absolute dependence on monovalent cations for activity, K⁺ being the principal activator. The phosphatase was inhibited non-selectively by monovalent cations. The total activity of pyruvate kinase and its concentration on a tissue fresh weight basis was greatest in the leaves, activity increasing with the maturity of the tissue. Less enzyme was present in roots, and least in the fruit.     

Mechanism of activation of pyruvate dehydrogenase by mitogens in pig lymphocytes.

The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.     

Insulin mediator causes dephosphorylation of the alpha subunit of pyruvate dehydrogenase by stimulating phosphatase activity

Insulin treatment of rats results in an increased amount or activity of insulin mediators in liver and skeletal muscle. These mediators stimulated pyruvate dehydrogenase and inhibited adenylate cyclase. The insulin-generated mediators caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in mitochondria prelabeled with [gamma-32P]ATP. An assay was developed which quantitatively measured mediator activity by determining the rate of alpha-subunit dephosphorylation. The dephosphorylation was directly proportional to the amount of mediator added and was directly related to activation of pyruvate dehydrogenase. The decrease of alpha-subunit phosphorylation resulted from stimulation of pyruvate dehydrogenase phosphatase, since it occurred in the absence of ATP and was inhibited by NaF. These data further delineate the mechanism of insulin mediator activation of pyruvate dehydrogenase.     

Involvement of Vitamin B6 in the Dethiomethylation of Methionine by Rumen Microorganisms

The dethiomethylation of methionine by a dialyzed extract obtained from the protozoa-rich fraction of rumen fluid is stimulated 2.5-fold by pyridoxal phosphate and strongly inhibited by deoxypyridoxine, a pyridoxal phosphate antagonist. These effects are not seen with undialyzed extracts or with whole rumen fluid. It is suggested that the anaerobic dethiomethylation of methionine by rumen microorganisms requires pyridoxal phosphate as a cofactor.