Glycolysis* and Gluconeogenesis**

Carbohydrate Metabolism

Select the human MAP for starch and sucrose metabolism with a colored map of human enzymes marked as green shaded boxes.

Carbohydrates are found as monosaccharides, disaccharides, and polysaccharides or complex carbohydrates. They function in energy storage (starch&glycogen), signaling (glycoproteins and glycolipids, e.g. blood group determinants), fuel the nervous system and muscle (and virtually all cells, although there are distinct cell type specific differences in choice of primary fuel molecule), are parts of nucleic acids (genes, mRNA, tRNA, ribosomes), and as cell surface markers and signaling in glycolipids and glycoproteins, are part of connective tissue (heteropolymers; glycosaminoglycans), cell wall components (cellulose, hemi-cellulose) made of polymers that are enzymatically inert for most mammals to digest (except ruminants that harbor a special digestive tract bacterium with the appropriate cellulase enzyme). The alpha glycosidic bonds found in glycogen and starch are metabolically available to humans.

Starch is the major source of dietary glucose. The enzymes responsible for starch degradation are called amylases. Other sources of glucose are sucrose, a disaccharide glucose-fructose from fruits, and lactose, a glucose-galactose disaccharide from milk. Only monosaccharide species like glucose, fructose and galactose can be absorbed via active membrane transport systems. Special intestinal glucosidases split the disaccharides into their monosaccharide components. Maltose is hydrolyzed by isomaltase (oligo-1,6-glucosidase, E.C. and, with lower efficacy, by sucrase (sucrose alpha-glucosidase, E.C. Lactose intolerance comes from a lack of lactase in many adults, causing an accumulation of milk sugar with consequences such as dehydration.

There is no specific carbohydrate requirement for humans except for Vitamin C, the C6 compound ascorbic acid. Man, some primates, and guinea pigs lack one of the enzymes required for the synthesis of L-ascorbic acid. This has been understood as an inborn metabolic disorder arising during primate evolution. Consequently, L-ascorbic acid has to be taken up with our food. All other monosaccharides can be synthesized and are not required dietary supplements.

Once the intestinal mucosal cell of the small intestine have transported the monosaccharides into the blood circulatory system they can pass directly into the liver, where fructose and galactose are converted into glucose. The principal role of the liver is to act as a blood glucostat. Excess glucose will be stored as glycogen mainly in liver and muscle cells or in form of metabolized fat in adipocytes. Only glycogen, but not fats, can later be metabolized when food intake is restricted to maintain an adequate level of glucose in the blood stream. Fat, however, can be used for the oxidative regeneration of ATP and reductive power (NADH).

(KEGG Pathway MAP00100)

Glucose metabolism is conserved throughout evolution, but species and tissue specific variations are well known and of physiological importance. The well studied metabolic pathways of glucose oxidation for energy usage are derived from biochemical studies of mammalian liver, muscle, and brain tissue as well as  E.coli. The major pathways are glycolysis and the pentose-phosphate pathway. producing pyruvate

Pathway Substrate Product Energy stored as
Glycolysis Glucose (1 mol) Pyruvate (2 mol) ATP & NADH (2 mol)
Pentose Phosphate Cycle Glucose (1 mol) CO2 (6 mol) NADPH (12 mol)

Glycolysis is used by both aerobic and anaerobic organisms. Glycolysis in human and bacteria are almost identical with respect to the enzymes employed, but differ by their uptake mechanism of glucose into the cell and the end product under anaerobic conditions. In humans glucose enters the cytoplasm through glucose facilitators (passive diffusion). In enteric bacteria glucose intake is fueled by concomitant phosphorylation, while the hexose is transported across the membrane.

Step Energy yield
1. Committed step of glucose phosphorylation to Glucose-6-phosphate - 1 ATP
2. Converting glucose-6-P into the ketose form fructose-6-phosphate
3. Phosphorylating fructose-6-phosphate to Fructose-1,6- bisphosphate - 1 ATP
4. Fructose-1,6-biphosphate is split into two chemically different trioses, glycerone-P (or dihydroxyacetone-P) and glyceraldehyde-3-P (GAP)
5. Glyceron-P (DHAP) is isomerized to GAP resulting in two metabolically equivalent glyceraldehyde-3-P
6. Glyceraldehyd-3-P oxidized and phosphorylated to glycerate-1,3-biphosphate + 2 NADH
7. Glycrate-1,3-biphosphate converted to 3-phosphoglycerate + 2 ATP
8. Phosphate at position C3 will now be moved to position C2 forming glycerate-2-P
9. Elimination reaction (-H2O) producing phosphoenolpyruvate
10. Phosphate will be transferred to form ATP and Pyruvate + 2 ATP
11. Pyruvate to lactate reduction (under anaerobic conditions; provides NAD+ for step 6) - 2 NADH

Note: At step 7 glycolysis has regained the phosphoryltransfer potential it invested in the first three steps to commit glucose to oxidative degradation. The net energy gained so far in form of ATP is zero, but the pathway yielded 2 mols of NADH. Up to here glucose phosphorylation served as catalyst to extract reducing power. Considering the intermediate of two mols of NADH, the net production for the conversion of one mol of glucose to pyruvate is thus 2 mols of ATP and 2 mols of NADH. This is the net energy yield of glycolysis under aerobic conditions.

Under anaerobic conditions, the reduction of pyruvate to lactate (C00186) by means of NADH oxidation is the only source of NAD+ for glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) in step 6. Depleting the cytoplasm of NAD+ effectively inhibits glycolysis. Lactate formation is the endpoint of glycolysis in mammals. The sustained use of skeletal muscle under stress is a typical anaerobic reaction and the accumulation of lactate is the cause of sour muscles after an unexpected, heavy work out.

In E.coli (and other microorganisms) the main products formed under anaerobic conditions are in decreasing order formate, ethanol, acetate, and succinate. While succinate is directly obtained from phosphoenolpyruvate, formate and acetate are produced from pyruvate. The third reaction step (shown below) is the major source of ATP under strict anaerobic conditions and pyruvate as the only energy source (no glucose) for this bacterium.

                                     pyruvate + CoA Û acetyl-CoA + formate

                                     acetyl-CoA + Pi Û acetyl-P + CoA

                                     acetyl-P + ADP Û acetate + ATP

(KEGG Pathway MAP00100)

The regeneration of glucose is called gluconeogenesis and is particularly important in liver, which is the major organ involved in glucose synthesis from carbohydrate and non-carbohydrate sources. It is the only organ that can regenerate glucose form lactate. Comparing glycolysis and its reversed mode gluconeogenesis demonstrates general rules governing metabolic pathways regarding control and reversibility. While 7 out of 10 glycolytic steps are reversible as they exhibit small change of free energy, three steps occur at a considerable larger free energy change (less than -4kcal/mol) making them irreversible. In gluconeogenesis the glycolytic enzymes are bypassed by gluconeogenetic enzymes.

Reaction Glycolytic enzyme Gluconeogenetic enzyme
Glucose Û glucose-6-phosphate Glucokinase Glucose-6-phosphatase
Fructose-6-P Û fructose-1,6-diP Phosphofructokinase Fructose-1,6-biphosphatase
Phosphoenolpyruvate Û pyruvate Pyruvate kinase

Pyruvate carboxylase
Phosphoenolpyruvate carboxykinase 1

Comparing the energy expenditure for gluconeogenesis with the gain from glycolysis clearly shows that it costs more to generate glucose from internal metabolites than to break it down once glucose is inside the cell:

1 Glucose Þ 2 Lactate + 2 ATP
2 Lactate + 6 ATP Þ 1 Glucose

Control of Glycolysis and Gluconeogenesis

In mammals the balance between gluconeogenesis and glycolysis in liver has to respond to the needs of the entire body such as mechanical work, digestion, or thinking and thus is carefully adjusted. Here is a list of internal and external molecules that affect the balance between glycolysis and gluconeogenesis.

Intracellular regulators include amino acids, the citric acid cycle intermediate citrate and the acyl intermediates acetyl-CoA and fatty acids. All will stimulate gluconeogenesis through a positive feed forward control of pyruvate carboxylase and fructose-1,6-biphosphatase, respectively, if they are abundant. In addition, the energy charge of a cell defined by the availability of ATP, ADP, AMP control glycolysis and gluconeogenesis (see respiratory control). Low energy levels (AMP, ADP) stimulate glycolysis, high energy levels (ATP) inhibit glycolysis and activate gluconeogenesis.

Hormones are extracellular regulators which also affect glycolysis and gluconeogenesis. They coordinate metabolic activity among different organs. Insulin stimulates glycolysis to lower blood glucose levels after a meal to signal energy availability to all organs, while glucagon and epinephrine (adrenaline) stimulate gluconeogenesis (and lipolysis in fatty tissue by hepatic lipase) to provide energy to muscle and brain during stress. Regulation includes enzyme modulation (phosphorylation) and enzyme biosynthesis (gene expression).

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