John F. Turner

Glucokinase of pea seeds

1. Glucokinase (ATP : D-glucose 6-phosphotransferase, EC 2.7.1.2) was extracted from pea seeds and purified by fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and Sephadex. 2. The relative rates of phosphorylation of glucose, mannose and fructose (final concentration 5 mM) were 100, 64 and 11. 3. The Km for glucose of pea-seed glucokinase was 70 muM and the Km for mannose was 0.5 mM. The Km for fructose was much higher (30 mM). 4. Mg2+ ions were essential for activity. Mn2+ could partially replace Mg2+. 5. Enzyme activity was not inhibited by glucose 6-phosphate. A number of other metabolites had no effect on glucokinase activity. 6. Pea-seed glucokinase was inhibited by relatively low concentrations of ADP.

PYRUVATE KINASE OF HIGHER PLANTS

Abstract 1. 1. Pyruvate kinase (EC 2.7.1.40) was extracted from pea seeds and carrots and partially purified by fractionation with (NH4)2SO4. 2. 2. The enzyme from both sources showed hyperbolic kinetics for the substrates phosphoenolpyruvate and ADP. Mg2+ or Mn2+ and a movement cation were required for enzyme activity. Ca2+ inhibited enzyme activity. 3. 3. Citrate and ATP inhibited the activity of both enyzme preparations. 4. 4. Fructose 1,6-diphosphate and a number of other metabolites and intermediates had no effect on pea-seed or carrot pyruvate kinase. 5. 5. Pyruvate kinase from a number of other plant tissues was not affected by the addition of fructose 1,6-diphosphate. 6. 6. The possible role of pyruvate kinase in the regulation of carbohydrate metabolism in plants is discussed.

The regulation of pea-seed phosphofructokinase by 6-phosphogluconate, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate.

1. 1. 6-Phosphogluconate, 3-phosphoglycerate and 2-phosphoglycerate were allosteric modifiers fo pea-sed phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) and each inhibited enzyme activity. The inhibitions were relieved by increasing the concentration of fructose 6-phosphate or by lowering the ATP concentration. In the presence of Pi, at low ATP concentrations, 6-phosphogluconate, 3-phosphoglycerate and 2-phosphoglycerate stimulated phosphofructokinase activity. 2. 2. Experiments with other intermediates of carbohydrate metabolism showed that 2,3-diphosphoglycerate inhibited pea-seed phosphofructokinase, but glucose 6-phosphate, pyruvate, succinate and malate had no effect. 3. 3. A common mechanism is suggested for the interaction of 6-phosphogluconate, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate with the enzyme molecule. 4. 4. The possible significance of these modifiers of pea-seed phosphofructokinase in the regulatory control of plant carbohydrate metabolism is discussed.

Cooperativity in pea-seed phosphofructokinase

Abstract 1. 1. The effects of concentration of substrates on the activity of pea-seed phosphofructokinase (ATP: d -fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) were examined by determining substrates saturation curves and replotting these according to the Hill equation. 2. 2. The curve of enzyme activity versus concentration of Fru-6- P contained two points of inflection and an intermediary plateau region. This curve was consistent with negative cooperative kinetics at lower Fru-6- P concentrations and positive cooperative kinetics at higher concentrations of Fru-6- P . 3. 3. The inhibitor phosphoenolpyruvate induced a change from negative cooperative kinetics to positive cooperative kinetics for Fru-6- P at low concentrations of this substrate. In the presence of the stimulator P i the positive cooperative kinetics for Fru-6- P at high concentrations were altered towards Michaelis-Menten kinetics. 4. 4. The interactions between pea-seed phosphofructokinase and the substrate MgATP 2− followed negative cooperative kinetics and were not influenced by either the stimulator P i or the inhibitor phosphoenolpyruvate. 5. 5. Possible explanations for these kinetic properties of pea-seed phosphofructokinase are considered and the relevance of the results to the possible regulatory role of the enzyme in plant carbohydrate metabolism is discussed.

4 – The Regulation of Glycolysis and the Pentose Phosphate Pathway

Publisher Summary This chapter discusses the regulation of glycolysis and the pentose phosphate pathway. Glycolysis and the pentose phosphate pathway are the two main pathways of carbohydrate degradation in plants. Sucrose and starch are the principal sources of substrates for glycolysis in plants. The enzymes involved in the breakdown of sucrose and starch in plants do not appear to have regulatory properties. The localization of metabolic pathways in subcellular compartments is an aspect of control that has significance for the regulation of carbohydrate metabolism in plants. The chapter presents compartmentation of the enzymes glycolysis. Glycolysis occurs in plastids and in the cytoplasm of plant tissues; the reactions in the respective compartments are catalyzed by separate isoenzymes. The relative amounts of plastid and cytoplasmic isoenzymes differ for the various glycolytic reactions and depend on the type of tissue and its stage of development.Publisher Summary This chapter discusses the regulation of glycolysis and the pentose phosphate pathway. Glycolysis and the pentose phosphate pathway are the two main pathways of carbohydrate degradation in plants. Sucrose and starch are the principal sources of substrates for glycolysis in plants. The enzymes involved in the breakdown of sucrose and starch in plants do not appear to have regulatory properties. The localization of metabolic pathways in subcellular compartments is an aspect of control that has significance for the regulation of carbohydrate metabolism in plants. The chapter presents compartmentation of the enzymes glycolysis. Glycolysis occurs in plastids and in the cytoplasm of plant tissues; the reactions in the respective compartments are catalyzed by separate isoenzymes. The relative amounts of plastid and cytoplasmic isoenzymes differ for the various glycolytic reactions and depend on the type of tissue and its stage of development.

Pea seed triose phosphate isomerase

Abstract Triose phosphate isomerase was purified ca 250-fold from pea seed extracts. The k m for D -glyceraldehyde-3-P was 0.44 mM and for dihydroxyacetone-P, 0.88 mM. P-enolpyruvate, 2-P-glycerate, 3-P-glycerate and 2-P-glycolate were strongly inhibitory. Pi and arsenate also inhibited pea seed triose phosphate isomerase.

Phosphofructokinase of carrot roots

Abstract Phosphofructokinase was partially purified from carrot root extracts. Monovalent cations stimulated carrot phosphofructokinase activity. The enzyme was strongly inhibited by P-enolpyruvate and this inhibition was relieved by NACl or KCl. Pi inhibited the enzyme at pH 7.9 but was stimulatory at pH 6.6.