Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L. (1/165)

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.  (+info)

A functional arginine residue in rabbit-muscle aldolase. (2/165)

Rabbit muscle aldolase is irreversibly modified by the arginine-selective alpha-dicarbonyl, phenylglyoxal, loss of activity correlating with the unique modifications of one arginine residue per subunit, as determined by amino acid analysis, and (7-14C)phenylglyoxal incorporation. The affinity of the modified enzyme for dihydroxyacetone phosphate is significantly reduced while substantial protection against inactivation is afforded by fructose 1,6-disphosphate, dihydroxyacetone phosphate or phosphate ion. The nature of the substrate C-1 phosphate binding site in this enzyme is discussed in the light of these and other results.  (+info)

Exploring substrate binding and discrimination in fructose1, 6-bisphosphate and tagatose 1,6-bisphosphate aldolases. (3/165)

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosphate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the transition state analogue phosphoglycolohydroxamate delineated the roles of individual amino acids in binding glycerone-P and in the initial proton abstraction steps of the mechanism. The X-ray structure has now been used, together with sequence alignments, site-directed mutagenesis and steady-state enzyme kinetics to extend these studies to map important residues in the binding of glyceraldehyde 3-phosphate. From these studies three residues (Asn35, Ser61 and Lys325) have been identified as important in catalysis. We show that mutation of Ser61 to alanine increases the Km value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this effect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on catalysis emphasizing the importance of an hydroxyl group for this role. Mutation of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this residue effects the binding of both triose substrates. Finally, mutation of Lys325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in maintaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no significant effect on the function of the enzyme. In a separate study to characterize the molecular basis of aldolase specificity, the agaY-encoded tagatose 1,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically characterized. Our studies showed that the two aldolases are highly discriminating between the diastereoisomers fructose bisphosphate and tagatose bisphosphate, each enzyme preferring its cognate substrate by a factor of 300-1500-fold. This produces an overall discrimination factor of almost 5 x 105 between the two enzymes. Using the X-ray structure of the fructose 1,6-bisphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the active site. These residues might potentially be important in substrate recognition. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagatose 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active site, many of which abolished fructose 1, 6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzyme catalysis in this family of enzymes, and points to the need for further structural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.  (+info)

Evidence for the coordinate control of glycogen synthesis, glucose utilization, and glycolysis in Escherichia coli. II. Quantitative correlation of the inhibition of glycogen synthesis and the stimulation of glucose utilization by 2,4-dinitrophenol with the effects on the cellular levels of glucose 6-phosphate, fructose, 1,6-diphosphate, and total adenylates. (4/165)

In cultures of Escherichia coli W4597(K) and G34 under various nutritional conditions the rates of glucose utilization and cellular levels of fructose-1,6-P2 are quantitatively related by the Hill equation where the value of the Hill coefficient is approximately equal to 2. This is the first evidence that fructose-P2, or any metabolite which covaries with fructose-P2, modulates glucose utilization in E. coli. In light of previous observations from our laboratory this new observation and those in the succeeding report provide the first evidence that in E. coli glycolsis, glycogen synthesis and glucose utilization are coordinately regulated, thus providing for the coupling of ATP utilization and production under various metabolic circumstances. Alterations in the level of ATP apparently affect the velocity of phosphofructokinase, the rate-limiting enzyme in glycolsis, altering the cellular levels of glucose-6-P or fructose-P2. Changes in the levels of these hexose phosphates are quantitatively related to alterations in the rates of glucose utilization and glycogen synthesis in the intact E. coli cell.  (+info)

Control of fructose and citrate synthesis in guinea pig seminal vesicle epithelium. (5/165)

Glucose utilization, biosynthesis of fructose and citrate, and certain aspects of energy metabolism were studied in a homogeneous preparation of mating guinea pig seminal vesicle epithelium. Under aerobic conditions, ATP:ADP ratios between 6 and 7 were maintained attesting to the viability and metabolic integrity of the preparation. There were multiple differences between seminal vesicle epithelium and smooth muscle on comparison of concentrations of 19 different metabolites including the adenine nucleotides. In seminal vesicle, glycolysis was rapid under anaerobic conditions (approximately 25 mumol times g-1 times hour-1 during the first 3 min) and also occurred under aerobic conditions (approimately 5 mumol times g-1 times hour-1). Anaerobically, the concentration of fructose diphosphate increased 2-fold and that of fructose 6-phosphate decreased to one-third of control values, consistent with regulation at the level of phosphofructokinase. ATP and total adenine nucleotides decreased rapidly and, by 3 min, had attained steady state values of about one-third and one-half of control values, respectively. Under aerobic conditions, the rate of fructogenesis increased with increasing concentrations of added glucose, reaching maximum (3 mumol times g-1 times hour-1) at 8 to 10 mM glucose and half-maximum at 2 mM glucose. Simultaneously, sorbitol synthesis occurred at rates that were similarly influenced by the concentrations of added glucose. The highest rate of fructogenesis (6.5 mumol times g-1 times hour-1) occurred during aerobic incubation immediately after a period of anaerobic incubation: exogenous substrate was not required. This could be prevented by addition of D-glyceraldehyde which was converted to glycerol stoichiometrically because of its preferential (versus glucose) reduction by aldose reductase. Our data are consistent with the sorbitol pathway of Hers as the major route of fructose biosynthesis. The rate of citrate syntehsis increased with increasing concentrations of added pyruvate. The maximum rate (3.4 mumol times g-1 times hour-1) was achieved with 2 mM pyruvate. Added glucose failed to support citrate synthesis to the same extent as did added pyruvate. The highest rate of citrate synthesis (8.0 mumol times g-1 times hour-1) occurred simultaneously with the highest rate of fructogenesis (after the anaerobic-aerobic transition). Exogenous substrate was not required.  (+info)

Renaturation of acid-denatured rabbit muscle aldolase. Existence and properties of a stable monomeric intermediate. (6/165)

The effects of temperature, pH and the substrate, fructose 1,6-bisphosphate, upon the kinetics and yield of renaturation of acid-denatured rabbit muscle aldolase have been investigated. The results are discussed in terms of a sequential set of events leading from the unfolded polypeptide chain to the renatured oligomeric enzyme. One of the intermediate molecular species in this sequence has been characterized as a folded monomer with a sedimentation coefficient of 3.1 S. This monomer is shown to be much more heat-labile than the tetramer under identical conditions, thus demonstrating stabilization of the tertiary structure of the polypeptide chain by the quaternary interactions between protomers.  (+info)

A comparative study on diurnal changes in metabolite levels in the leaves of three crassulacean acid metabolism (CAM) species, Ananas comosus, Kalanchoe daigremontiana and K. pinnata. (7/165)

A comparative study on diurnal changes in metabolite levels associated with crassulacean acid metabolism (CAM) in the leaves of three CAM species, Ananas comosus (pineapple), a hexose-utilizing species, and Kalanchoe daigremontiana and K. pinnata, two starch-utilizing species, were made. All three CAM species showed a typical feature of CAM with nocturnal malate increase. In the two Kalanchoe species, isocitrate levels were higher than citrate levels; the reverse was the case in pineapple. In the two Kalanchoe species, a small nocturnal citrate increase was found and K. daigremontiana showed a small nocturnal isocitrate increase. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (F-6-P) and glucose 1-phosphate (G-1-P) levels in the three CAM species rose rapidly during the first part of the dark period and decreased during the latter part of the dark period. The levels of the metabolites also decreased during the first 3 h of the light period, then, remained little changed through the rest of the light period. Absolute levels of G-6-P, F-6-P and G-1-P were higher in pineapple than in the two Kalanchoe species. Fructose 1,6-bisphosphate (F-1,6-P(2)) levels in the three CAM species increased during the dark period, then dramatically decreased during the first 3 h of the light period and remained unchanged through the rest of the light period. The extent of nocturnal F-1,6-P(2) increase was far greater in the two Kalanchoe species than in pineapple. Absolute levels of F-1,6-P(2) were higher in the two Kalanchoe species than in pineapple, especially during dark period. Diurnal changes in oxaloacetate (OAA), pyruvate (Pyr) and phosphoenolpyruvate (PEP) levels in the three CAM species were similar.  (+info)

Control of phosphofructokinase from rat skeletal muscle. Effects of fructose diphosphate, AMP, ATP, and citrate. (8/165)

Under conditions used previously for demonstrating glycolytic oscillations in muscle extracts (pH 6.65, 0.1 to 0.5 mM ATP), phosphofructokinase from rat skeletal muscle is strongly activated by micromolar concentrations of fructose diphosphate. The activation is dependent on the presence of AMP. Activation by fructose diphosphate and AMP, and inhibition by ATP, is primarily due to large changes in the apparent affinity of the enzyme for the substrate fructose 6-phosphate. These control properties can account for the generation of glycolytic oscillations. The enzyme was also studied under conditions approximating the metabolite contents of skeletal muscle in vivo (pH 7.0, 10mM ATP, 0.1 mM fructose 6-phosphate). Under these more inhibitory conditions, phosphofructokinase is strongly activated by low concentrations of fructose diphosphate, with half-maximal activation at about 10 muM. Citrate is a potent inhibitor at physiological concentrations, whereas AMP is a strong activator. Both AMP and citrate affect the maximum velocity and have little effect on affinity of the enzyme for fructose diphosphate.  (+info)

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