Values For Phosphoenolpyruvate Research Articles

Pyruvate kinase 2 from Synechocystis sp. PCC 6803 increased substrate affinity via glucose-6-phosphate and ribose-5-phosphate for phosphoenolpyruvate consumption

Pyruvate kinase (Pyk, EC 2.7.1.40) is a glycolytic enzyme that generates pyruvate and adenosine triphosphate (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP), respectively. Pyk couples pyruvate and tricarboxylic acid metabolisms. Synechocystis sp. PCC 6803 possesses two pyk genes (encoded pyk1, sll0587 and pyk2, sll1275). A previous study suggested that pyk2 and not pyk1 is essential for cell viability; however, its biochemical analysis is yet to be performed. Herein, we biochemically analyzed Synechocystis Pyk2 (hereafter, SyPyk2). The optimum pH and temperature of SyPyk2 were 7.0 and 55 °C, respectively, and the Km values for PEP and ADP under optimal conditions were 1.5 and 0.053 mM, respectively. SyPyk2 is activated in the presence of glucose-6-phosphate (G6P) and ribose-5-phosphate (R5P); however, it remains unaltered in the presence of adenosine monophosphate (AMP) or fructose-1,6-bisphosphate. These results indicate that SyPyk2 is classified as PykA type rather than PykF, stimulated by sugar monophosphates, such as G6P and R5P, but not by AMP. SyPyk2, considering substrate affinity and effectors, can play pivotal roles in sugar catabolism under nonphotosynthetic conditions.

Kinetic Characterization and Inhibitor Screening of Pyruvate Kinase I From Babesia microti.

The apicomplexan Babesia microti is a main pathogenic parasite causing human babesiosis, which is one of the most widely distributed tick-borne diseases in humans. Pyruvate kinase (PYK) plays a central metabolic regulatory role in most living organisms and catalyzes the essentially irreversible step in glycolysis that converts phosphoenolpyruvate (PEP) to pyruvate. Hence, PYK is recognized as an attractive therapeutic target in cancer and human pathogens such as apicomplexans. In this study, we cloned, expressed, and purified B. microti PYK I (BmPYKI). Western blotting illustrated that anti-rBmPYKI antibody could specifically recognize the native BmPYKI protein in the lysate of B. microti with a 54-kDa band, which is consistent with the predicted size. In addition, the enzymatic activity of the purified recombinant PYKI (rPYKI) was tested under a range of pH values. The results showed that the maximum catalytic activity could be achieved at pH 7.0. The saturation curves for substrates demonstrated that the Km value for PEP was 0.655 ± 0.117 mM and that for ADP was 0.388 ± 0.087 mM. We further investigated the effect of 13 compounds on rBmPYKI. Kinetic analysis indicated that six inhibitors (tannic acid, shikonin, apigenin, PKM2 inhibitor, rosiglitazone, and pioglitazone) could significantly inhibit the catalytic activity of PYKI, among which tannic acid is the most efficient inhibitor with an IC50 value 0.49 μM. Besides, four inhibitors (tannic acid, apigenin, shikonin, and PKM2 inhibitor) could significantly decrease the growth of in vitro-cultured B. microti with IC50 values of 0.77, 2.10, 1.73, and 1.15 μM. Overall, the present study provides a theoretical basis for the design and development of new anti-Babesia drugs.

Kinetic and molecular characterization of the pyruvate phosphate dikinase from Trypanosoma cruzi

Trypanosoma cruzi, like other trypanosomatids analyzed so far, can use both glucose and amino acids as carbon and energy source. In these parasites, glycolysis is compartmentalized in glycosomes, authentic but specialized peroxisomes. The major part of this pathway, as well as a two-branched glycolytic auxiliary system, are present in these organelles. The first enzyme of one branch of this auxiliary system is the PPi-dependent pyruvate phosphate dikinase (PPDK) that converts phosphoenolpyruvate (PEP), inorganic pyrophosphate (PPi) and AMP into pyruvate, inorganic phosphate (Pi) and ATP, thus contributing to the ATP/ADP balance within the glycosomes. In this work we cloned, expressed and purified the T. cruzi PPDK. It kinetic parameters were determined, finding KM values for PEP, PPi and AMP of 320, 70 and 17 μM, respectively. Using molecular exclusion chromatography, two native forms of the enzyme were found with estimated molecular weights of 200 and 100 kDa, corresponding to a homodimer and monomer, respectively. It was established that T. cruzi PPDK's specific activity can be enhanced up to 2.6 times by the presence of ammonium in the assay mixture. During growth of epimastigotes in batch culture an apparent decrease in the specific activity of PPDK was observed. However, when its activity is normalized for the presence of ammonium in the medium, no significant modification of the enzyme activity per cell in time was found.

Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase

This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj_PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj_PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj_PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn2+, and HCO3−, being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn2+. Therefore in Lmj_PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj_PEPCK

Stereochemistry of the carboxylation reaction catalyzed by the ATP-dependent phosphoenolpyruvate carboxykinases from Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens

The stereochemistry of CO 2 addition to phosphoenolpyruvate (PEP) to yield oxaloacetate catalyzed by ATP-dependent Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens PEP carboxykinases was determined using (Z)-3-fluorophosphoenolpyruvate ((Z)-F-PEP) as a substrate analog. A. succiniciproducens and S. cerevisiae PEP carboxykinases utilized (Z)-F-PEP with 1/14 and 1/47 the respective K m values for PEP. On the other hand, in the bacterial and yeast enzymes k cat was reduced to 1/67 and 1/48 the value with PEP, respectively. The binding affinity of pyridoxylphosphate-labeled S. cerevisiae and A. succiniciproducens PEP carboxykinases for PEP and (Z)-F-PEP was checked and found to be of similar magnitude for both substrates, suggesting that the lowered K m values for the fluorine-containing PEP analog are due to kinetic effects. The lowered k cat values when using (Z)-F-PEP as substrate suggest that the electron withdrawing effect of fluorine affects the nucleophilic attack of the double bond of (Z)-F-PEP to CO 2. For the stereochemical analyses, the carboxylation of (Z)-F-PEP was coupled to malate dehydrogenase to yield 3-fluoromalate, which was analyzed by 19F NMR. The fluoromalate obtained was identified as (2 R, 3 R)-3-fluoromalate for both the A. succiniciproducens and S. cerevisiae PEP carboxykinases, thus indicating that CO 2 addition to (Z)-F-PEP, and hence PEP, takes place through the 2- si face of the double bond. These results, together with previously published data [Rose, I.A. et al. J. Biol. Chem. 244 (1969) 6130–6133; Hwang, S.H. and Nowak, T. Biochemistry 25 (1986) 5590–5595] indicate that PEP carboxykinases, no matter their nucleotide specificity, catalyze the carboxylation of PEP from the 2- si face of the double bond.

Inhibitory effect of phosphoenolpyruvate on glycolytic enzymes in Escherichia coli

For analyzing the control of energy metabolism in Escherichia coli, we carried out kinetic analyses of glycolytic enzymes purified from the overexpressing clones of E. coli K12 W3110 that were constructed with the vector pCA24N. Phosphoenolpyruvate (PEP) acted as an effective inhibitor of enzymes of the preparatory phase in glycolysis. Glucokinase was potently inhibited by PEP in a competitive manner with respect to ATP: the K i value for PEP was 0.1 mM. PEP further inhibited phosphoglucoisomerase to a lesser extent, and phosphofructokinase A and aldolase A with 10-fold the K i values of glucokinase and phosphoglucoisomerase. Glucose is incorporated into E. coli through two pathways: the PTS (PEP-dependent phosphotransferase system) and the glucokinase reaction. PEP, a potent inhibitor of E. coli glucokinase, unlike most eukaryotic hexokinases, can act as a signal molecule controlling glucose uptake and glycolytic flux in cells.

Roles of Asp75, Asp78, and Glu83 of GTP-dependent Phosphoenolpyruvate Carboxykinase from Mycobacterium smegmatis

The roles of Asp(75), Asp(78), and Glu(83) of the (75)DPSDVARVE(83) element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp(78) and Glu(83) are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp(78) influences Tyr(220); Tyr(220) helps to position bound PEP, and Glu(83) interacts with Arg(81). Experimental data on other PEPCKs indicate that Arg(81) binds PEP, and the phosphate of PEP interacts with Mn(2+) of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent K(m) value for Mn(2+) 170-fold. In contrast, Asp(75) is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, V(max) decreased 1.4-4-fold. K(m) values for PEP and Mn(2+) increased 3-9- and 1.2-10-fold, respectively. K(m) values for GDP and bicarbonate changed little. 2) For PEP formation, V(max) increased 1.5-2.7-fold. K(m) values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu(83)-Arg(81) interaction affected Arg(81)-PEP association. D78A change altered the Tyr(220)-PEP interaction. These events perturbed PEP-Mn(2+) interaction and consequently affected catalysis severely. In contrast, substitutions at Asp(75), a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp(75) substitutions affected PEP-Mn(2+) interaction by changing the positions of Asp(78), Arg(81), and Glu(83), which translated to differential effects on two directions.

A GTP-dependent Vertebrate-type Phosphoenolpyruvate Carboxykinase from Mycobacterium smegmatis

This is the first report on a bacterial verterbrate-type GTP-dependent phosphoenolpyruvate carboxykinase (PCK). The pck gene of Mycobacterium smegmatis was cloned. The recombinant PCK was overexpressed in Escherichia coli in a soluble form and with high activity. The purified enzyme was found to be monomeric (72 kDa), thermophilic (optimum temperature, 70 degrees C), very stable upon storage at 4 degrees C, stimulated by thiol-containing reducing agents, and inhibited by oxalate and by alpha-ketoglutarate. The requirement for a divalent cation for activity was fulfilled best by Mn(2+) and Co(2+) and poorly by Mg(2+). At 37 degrees C, the highest V(m) value (32.5 units/mg) was recorded with Mn(2+) and in the presence of 37 mm dithiothreitol (DTT). The presence of Mg(2+) (2 mm) greatly lowered the apparent K(m) values for Mn(2+) (by 144-fold in the presence of DTT and by 9.4-fold in the absence of DTT) and Co(2+) (by 230-fold). In the absence of DTT but in the presence of Mg(2+) (2 mm) as the co-divalent cation, Co(2+) was 21-fold more efficient than Mn(2+). For producing oxaloacetate, the enzyme utilized both GDP and IDP; ADP served very poorly. The apparent K(m) values for phosphoenolpyruvate, GDP, and bicarbonate were >100, 66, and 8300 micrometer, respectively, whereas those for GTP and oxaloacetate (for the phosphoenolpyruvate formation activity) were 13 and 12 microm, respectively. Thus, this enzyme preferred the gluconeogenesis/glycerogenesis direction. This property fits the suggestion that in M. smegmatis, pyruvate carboxylase is not anaplerotic but rather gluconeogenic (Mukhopadhyay, B., and Purwantini, E. (2000) Biochim. Biophys. Acta. 1475, 191-206). Both in primary structure and kinetic properties, the mycobacterial PCK was very similar to its vertebrate-liver counterparts and thus could serve as a model for these enzymes; examples for several immediate targets are presented.

Purification and characterization of phosphoenolpyruvate carboxykinase from the anaerobic ruminal bacterium Ruminococcus flavefaciens.

Phosphoenolpyruvate (PEP) carboxykinase was purified 42-fold with a 25% yield from cell extracts of Ruminococcus flavefaciens by ammonium sulfate precipitation, preparative isoelectric focusing, and removal of carrier ampholytes by chromatography. The enzyme had a subunit molecular mass of approximately 66.3 kDa (determined by mass spectrometry), but was retained by a filter having a 100-kDa nominal molecular mass cutoff. Optimal activity required activation of the enzyme by Mn2+ and stabilization of the nucleotide substrate by Mg2+. GDP was a more effective phosphoryl acceptor than ADP, while IDP was not utilized. Under optimal conditions the measured activity in the direction of PEP carboxylation was 17.2 micromol min-1 (mg enzyme)-1. The apparent Km values for PEP (0.3 mM) and GDP (2.0 mM) were 9- and 14-fold lower than the apparent Km values for the substrates of the back reaction (oxaloacetate and GTP, respectively). The data are consistent with the involvement of PEP carboxykinase as the primary carboxylation enzyme in the fermentation of cellulose to succinate by this bacterium.

Comparative studies of pyruvate kinase from PSE and normal pig muscles.

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with the enzyme from normal muscle, pyruvate kinase isolated from PSE muscle shows a five times lower Michaelis constant, Km, for phosphoenol pyruvate and a more than ten times higher Kcat/Km value. The pH dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lower Km value and a 100-fold increased kcat/Km value for phosphoenol pyruvate as compared to isoform 1. The presence of isoform 3 in PSE muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE muscles.

Interaction of bovine heart pyruvate kinase with phospholipids

The interaction between bovine heart pyruvate kinase and liposomes was investigated for various phospholipids as function of pH, and salt concentration using steady-state kinetics and ultracentrifugation. Liposomes made from erythrocyte total lipid fraction and individual phospholipids were used. Pyruvate kinase specific activity increases upon the interaction with the phospholipids. The activation is specifically sensitive to presence of phosphatidylserine in liposomes. l-serine, and phospho- l-serine which are main components of phosphatidylserine head group show also some activation effect. Efficient adsorption of pyruvate kinase to phosphatidylserine liposomes occurs in the pH range 6.0–8.0 and at low ionic strength. Interaction with phosphatidylserine liposomes results in the change of V max d and K m values for phospho enolpyruvate without marked effect on K m value for ADP, and Hill coefficients for both substrates. e The interaction does not seem to influence the cooperativity between binding sites.

Catalytic Role of an Arginine Residue in the Highly Conserved and Unique Sequence of Phosphoenolpyruvate Carboxylase1

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] has a highly conserved and unique sequence, 578-FHGRGGSIGRGGAP-591 (on Escherichia coli, PEPC), in which a GRGG motif is repeated twice with two intervening residues. Since previous chemical modification studies suggested the functional importance of arginine residues, the invariant Arg587 in this region was replaced with Ser, and the enzymatic properties of the resulting mutant enzyme (R587S) were investigated. Replacement led to virtual loss of the catalytic activity to form oxaloacetate. The specific activity was 37 nmol.min-1.mg-1, which corresponds to 2 x 10(-4)-fold the activity of the wild-type enzyme. However, the activity of bicarbonate- and Mg(2+)-dependent hydrolysis of phosphoenolpyruvate (PEP) to pyruvate appeared for the mutant enzyme with a specific activity of 2.1 mumol.min-1.mg-1. In view of the stepwise reaction mechanism proposed for PEPC, this activity can be attributed to impairment of the subsequent partial reaction(s) following the formation of the intermediate carboxyphosphate. The half-saturation concentration (S0.5) of HCO3- in R587S was about 100-fold that in the wild-type enzyme, whereas the respective values for PEP and Mg2+ were 20- and 15-fold, indicative of this residue participating in the binding of HCO3-.

Molecular and physiological evaluation of transgenic tobacco plants expressing a maize phosphoenolpyruvate carboxylase gene under the control of the cauliflower mosaic virus 35S promoter

The expression of maize (Zea mays) phosphoenolpyruvate carboxylase (PEPC) gene constructs in transgenic tobacco plants (Nicotiana tabacum) was studied. Where transcription was under the control of a CaMV 35S promoter, maize PEPC transcripts of the correct size were detected. Western blot analysis indicated that the transgenic plants contained about twice as much PEPC as non-transformed plants. Furthermore, the enzymatic activity of PEPC in the leaves of these transgenic plants was up to twice as high as that in non-transformed plants. Two forms of PEPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form (the maize isoform) gave a high apparentK m value for phosphoenolpyruvate (PEP) and a high maximum activity, and the other (the tobacco isoform) exhibited a low apparentK m value for PEP and a low maximum activity. These biochemical differences resulted in several significant physiological changes in the transgenic plants: (1) the growth rate of the transgenic plants was lower than that of non-transgenic plants: (2) chlorophyll content per leaf area was relatively lower in the transgenic plants; and (3) the quantum yield of photosynthesis in the transgenic plants was not affected by changes in leaf temperature.

Activation of Cytosolic Pyruvate Kinase by Polyethylene Glycol.

Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo.

PURIFICATION AND CHARACTERIZATION OF PYRUVATE KINASE FROM THE GREEN ALGA CHLAMYDOMONAS REINHARDTII1

ABSTRACTA single form of pyruvate kinase was isolated from the green alga Chlamydomonas reinhardtii Dang. (Chlorophyta) and partially purified over twentyfold, yielding a final specific activity of 2.68 μmol pyruvate produced‐min‐1.mg‐1 protein. Studies of its physical characteristics reveal that the pyruvate kinase is heat stable, is partially inactivated by sulfhydryl reagent N‐ethylmaleimide, and has a pH optimum at 6.8 and a native molecular mass of 224 kDa. Immunological precipitation and western blotting, using antibodies raised against Selenastrum minutum Naeg. (Chlorophyta) cytosolic pyruvate kinase, reveal that C. reinhardtii pyruvate kinase possesses a subunit molecular mass of 57 kDa, indicating a homo‐tetrameric structure. This enzyme exhibits an absolute requirement for a divalent cation that can be fulfilled, by Mg2+. The monovalent cation K+ acts as a strong activator. The Km values for phosphoenolpyruvate and adenosine diphosphate (ADP) are 0.16 mM and 0.18 mM, respectively. The enzyme is capable of using other nucleotides with Vmax for UDP, GDP, IDP, and CDP of 70%, 55%, 53%, and 25% of that with ADP, respectively. Dihydroxyacetone phosphate, ribulose 1,5‐bisphosphate, adenosine monophosphate (AMP), ribose‐5‐phosphate, and glyceraldehyde‐3‐phosphate are activators, whereas glutamate, orthophosphate, adenosine triphosphate (ATP), citrate, isocitrate, malate, oxalate, phosphoglycolate, and 2,3‐diphosphoglycerate are potent inhibitors of this enzyme. Dihydroxyacetone phosphate can reverse the inhibition by glutamate and phosphate. These properties are discussed in light of pyruvate kinase regulation during anabolic and catabolic respiration. Substrate interaction and product inhibition studies indicate that ADP is the first substrate bound to the enzyme and pyruvate is the last product released (Ordered Bi Bi mechanism).

Substrate synergism and the steady-state kinetic reaction mechanism for EPSP synthase from Escherichia coli.

Previous studies of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) have suggested that the kinetic reaction mechanism for this enzyme in the forward direction is equilibrium ordered with shikimate 3-phosphate (S3P) binding first followed by phosphoenolpyruvate (PEP). Recent results from this laboratory, however, measuring direct binding of PEP and PEP analogues to free EPSPS suggest more random character to the enzyme. Steady-state kinetic and spectroscopic studies presented here indicate that E. coli EPSPS does indeed follow a random kinetic mechanism. Initial velocity studies with S3P and PEP show competitive substrate inhibition by PEP added to a normal intersecting pattern. Substrate inhibition is proposed to occur by competitive binding of PEP at the S3P site [Ki(PEP) = 6-8 mM]. To test for a productive EPSPS.PEP binary complex, the reaction order of EPSPS was evaluated with shikimic acid and PEP as substrates. The mechanism for this reaction is equilibrium ordered with PEP binding first giving a Kia value for PEP in agreement with the independently measured Kd of 0.39 mM (shikimate Km = 25 mM). Results from this study also show that the 3-phosphate moiety of S3P offers 8.7 kcal/mol in binding energy versus a hydroxyl in this position. Over 60% of this binding energy is expressed in binding of substrate to enzyme rather than toward increasing kcat. Glyphosate inhibition of shikimate turnover was poor with approximately 8 x 10(4) loss in binding capacity compared to the normal reaction, consistent with the independently measured Kd of 12 mM for the EPSPS.glyphosate binary complex. The EPSPS.glyphosate complex induces shikimate binding, however, by a factor of 7 greater than EPSPS.PEP. Carboxyallenyl phosphate and (Z)-3-fluoro-PEP were found to be strong inhibitors of the enzyme that have surprising affinity for the S3P binding domain in addition to the PEP site as measured both kinetically and by direct observation with 31P NMR. The collective data indicate that the true kinetic mechanism for EPSPS in the forward direction is random with synergistic binding occurring between substrates and inhibitors. The synergism explains how the mechanism can be random with S3P and PEP, but yet equilibrium ordered with PEP binding first for shikimate turnover. Synergism also accounts for how glyphosate can be a strong inhibitor of the normal reaction, but poor versus shikimate turnover.

The effects of aestivation on the catalytic and regulatory properties of pyruvate kinase from Helix aspersa

1. 1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. 2. At pH 7.0 the apparent K m values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/l for the enzyme from mantle; those for ADP were 0.35 mmol/l for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. 3. Both enzymes were inhibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S 0.5 of 1.2 mmol/l, but had a markedly higher affinity for PEP, S 0.5 = 0.20 mmol/l during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.

Influence of Hormones, Second Messengers and pH on the Expression of Metabolic Responses to Anoxia in a Marine Whelk

ABSTRACT The roles of hormones, second messengers and pH in triggering or potentiating biochemical responses to anaerobiosis were evaluated using in vitro incubations of isolated muscle tissues (foot, radular retractor, ventricle) from the marine whelk Busycon canaliculatum (L.). Incubating tissues in vitro under anoxic conditions stimulated changes in muscle fructose-2,6-bisphosphate levels and pyruvate kinase kinetics (Km values for phosphoenolpyruvate, values for L-alanine) that were virtually equivalent to those that occur in vivo. Additions of hormones (epinephrine, norepinephrine, octopamine, serotonin, glucagon, insulin) or inhibitors of prostaglandin synthesis (dexamethasone, aspirin) had no effect on these metabolic responses to anoxia. The second messenger compounds, dibutyryl cyclic AMP and Ca2+ + ionophore A23187 + phorbol myristate acetate, produced isolated and tissue-specific responses in muscles incubated under aerobic conditions, but the magnitude and pattern of these responses differed from those seen in anoxia. Second messengers also had no effect on the development of biochemical responses in anoxic muscles. Tissue pH was artificially altered in order to evaluate the role of pH change (acidification occurs during anoxia in vivo) in the control of metabolic responses to anoxia. In all cases, the changes in the kinetic properties of pyruvate kinase (PK) correlated with the state of oxygenation of the tissue and not with the measured tissue pH value; higher tissue pH did not prevent anoxia-induced phosphorylation of PK and lower tissue pH did not alter the kinetic patterns of the aerobic enzyme. Overall, the study indicates that cells and tissues of the whelk respond individually to anoxia and that coordination of the action of protein kinases during anoxia is not mediated by pH or by common second-messenger mechanisms.

Purification and properties of DAHP synthase from Nocardia mediterranei

3-Deoxy- d- arabino-heptulosonate-7-phosphate (DAHP) synthase, the first enzyme of the shilimate pathway was isolated from Nocardia mediterranci. It has a molecular weight of approx, 135 000, and four identical subunits, each with a molecular weight of 35 000. The K m values for phospho enolpyruvate (PEP) and d-erythrose 4-phosphate (E-4- P) were 0.4 and 0.25 mM, respectively, and kinetic study showed that lTrp inhibited DAHP synthase activity, but was not competitive with respect to PEP orE-4- P. The enzyme activity was inhibited by excess ofE-4- P added in the incubation system. d-ribose 5-phosphate (R-5- P), d-glucose 6-phosphate (G-6- P) or d-sedoheptulose 7-phosphate (Su-7- P) etc. inhibited DAHP synthase in cell-free extract, but on partially purified enzyme no inhibitory effect was detected. The indirect inhibition ofR-5- P and other sugar phosphates was considered to be due to the formation ofE-4- P catalyzed by the related enzymes present in cell-free extract.

Regulation of aromatic amino acid biosynthesis in the ribulose monophosphate cycle methylotroph Nocardia sp. 239

The regulation of aromatic amino acid biosynthesis in Nocardia sp. 239 was studied. In cell-free extracts 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase activity was inhibited in a cumulative manner by tryptophan, phenylalanine and tyrosine. Chorismate mutase was inhibited by both phenylalanine and tyrosine, whereas prephenate dehydratase was very sensitive to inhibition by phenylalanine. Tyrosine was a strong activator of the latter enzyme, whereas anthranilate synthase was inhibited effectively by tryptophan. No clear repression of the synthesis of these enzymes was observed during growth of the organism in the presence of the aromatic amino acids. It is therefore concluded that in Nocardia sp. 239 synthesis of these amino acids is mainly regulated by feedback inhibition. The molecular organization and kinetic properties of DAHP synthase were studied in more detail following its purification. The molecular weight of the native enzyme and its single subunit species were estimated to be 168,000 and 41,000, respectively, suggesting that the enzyme is a tetramer. Apparent Km values for phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) were 45 and 370 µM, respectively. Tryptophan, phenylalanine and tyrosine inhibited DAHP synthase in a competitive manner with respect to E4P, with apparent Ki values of 3, 160 and 180 µM, respectively. In addition, tryptophan and E4P (apparent Ki values of 11 and 530 µM, respectively) were found to exert an uncompetitive and competitive inhibition, respectively, towards PEP.