Scientific investigations into fermentation of grape sugar were pioneering studies of glycolysis

The Fate of Pyruvate The sequence of reactions from glucose to pyruvate is similar in most organisms and most types of cells. The fate of pyruvate is variable. Three reactions of pyruvate are of prime importance: 1. Aerobic conditions: oxidation to acetyl CoA which enters the citric acid cycle for further oxidation 2. Anaerobic conditions (muscles, red blood cells): conversion to lactate 3. Anaerobic conditions (microorganisms, yeast): conversion to ethanol

Diverse Fates of Pyruvate

Metabolism of Pyruvate to Ethanol Ethanol is formed from pyruvate in yeast and several other microorganisms in anaerobic conditions. Two reactions required: The first step is the decarboxylation of pyruvate to acetaldehyde. Enzyme - pyruvate decarboxylase. Coenzyme - thiamine pyrophosphate (derivative of the vitamin thiamine B1) The second step is the reduction of acetaldehyde to ethanol. Enzyme - alcohol dehydrogenase (active site contains a zinc). Coenzyme – NADH.

The conversion of glucose into ethanol is an example of alcoholic fermentation. The net result of alcoholic fermentation is: Glucose+2Pi + 2ADP + 2H+  2 ethanol + 2CO2 + 2ATP + 2H2O The ethanol formed in alcoholic fermentation provides a key ingredient for brewing and winemaking. There is no net NADH formation in the conversion of glucose into ethanol. NADH generated by the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of acetaldehyde to ethanol.

Metabolism of Pyruvate to Lactate Lactate is formed from pyruvate in an animal organism and in a variety of microorganisms in anaerobic conditions. The conversion of glucose into lactate is called lactic acid fermentation. Enzyme - lactate dehydrogenase. Coenzyme – NADH.

Muscles of higher organisms and humans lack pyruvate decarboxylase and cannot produce ethanol from pyruvate Muscle contain lactate dehydrogenase. During intense activity when the amount of oxygen is limiting the lactic acid can be accumulated in muscles (lactic acidosis). Lactate formed in skeletal muscles during exercise is transported to the liver. Liver lactate dehydrogenase can reconvert lactate to pyruvate. Overall reaction in the conversion of glucose into lactate: Glucose + 2 Pi + 2 ADP  2 lactate + 2 ATP + 2 H2O As in alcoholic fermentation, there is no net NADH formation. NADH formed in the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of pyruvate.

Metabolism of Pyruvate to Acetyl CoA In aerobic conditions pyruvate is converted to acetyl coenzyme A (acetyl CoA). Acetyl CoA enters citric acid cycle where degrades to CO2 and H2O and the energy released during such oxidation is utilized in NADH and FADH2. Pyruvate is converted to acetyl CoA in the matrix of mitochondria. The overall reaction: Pyruvate + NAD+ + CoA  acetyl CoA + CO2 + NADH Reaction is catalyzed by the pyruvate dehydrogenase complex (three enzymes and five coenzymes). If pyruvate is converted to acetyl CoA, NADH formed in the oxidation of glyceraldehyde 3-phosphate ultimately transfers its electrons to O2 through the electron-transport chain in mitochondria.

Other Sugars Can Enter Glycolysis Glucose is the main metabolic fuel in most organisms Other sugars convert to glycolytic intermediates Fructose and sucrose (contains fructose) are major sweeteners in many foods and beverages Galactose from milk lactose (a disaccharide) Mannose from dietary polysaccharides, glycoproteins

The Entry of Fructose into Glycolysis Much of the ingested fructose is metabolized by the liver, using the fructose 1-phosphate pathway. The first step is the phosphorylation of fructose to fructose 1-phosphate by fructokinase. Fructose 1-phosphate is then split into glyceraldehyde and dihydroxyacetone phosphate, an intermediate in glycolysis, by a specific fructose 1 -phosphate aldolase. Glyceraldehyde is then phosphorylated to glyceraldehyde 3-phosphate, a glycolytic intermediate, by triose kinase.

Fructose Is Converted to Glyceraldehyde 3-Phosphate

Fructose can be phosphorylated to fructose 6-phosphate by hexokinase. However, the affinity of hexokinase for glucose is 20 times as great as it is for fructose. Little fructose 6-phosphate is formed in the liver because glucose is so much more abundant in this organ. Glucose, as the preferred fuel, is also trapped in the muscle by the hexokinase reaction. Because liver and muscle phosphorylate glucose rather than fructose, adipose tissue is exposed to more fructose than glucose. Hence, the formation of fructose 6-phosphate in the adipose tissue is not competitively inhibited to a biologically significant extent, and most of the fructose in adipose tissue is metabolized through fructose 6-phosphate.

The Entry of Galactose into Glycolysis Galactose is converted into glucose 6-phosphate in four steps. The first reaction is the phosphorylation of galactose to galactose 1-phosphate by galactokinase.

Galactose 1-phosphate react with uridine diphosphate glucose (UDP-glucose). UDP-galactose and glucose 1-phosphate are formed. Enzyme - galactose 1-phosphate uridyl transferase. The galactose moiety of UDP-galactose is then epimerized to glucose. The configuration of the hydroxyl group at carbon 4 is inverted by UDP-galactose 4-epimerase.

Glucose 1-phosphate, formed from galactose, is isomerized to glucose 6-phosphate by phosphoglucomutase.

The Entry of Mannose into Glycolysis Mannose is converted to Fructose 6-Phosphate in two steps. Hexokinase catalyzes the convertion of mannose into mannose 6-phosphate. Isomerase converts mannose 6-phosphate into fructose 6-phosphate (metabolite of glycolysis).

Intolerance to Milk Many people are unable to metabolize the milk sugar lactose and experience gastro-intestinal disturbances if they drink milk. Lactose intolerance, or hypolactasia, is caused by a deficiency of the enzyme lactase, which cleaves lactose into glucose and galactose. Microorganisms in the colon ferment undigested lactose to lactic acid generating methane (CH4) and hydrogen gas (H2). The gas produced creates the uncomfortable feeling of gut distention and the annoying problem of flatulence. The lactic acid is osmotically active and draws water into the intestine, as does any undigested lactose, resulting in diarrhea. The gas and diarrhea hinder the absorption of other nutrients (fats and proteins). Treatment: - to avoid the products containing lactose; - the enzyme lactase can be ingested.

Galactosemia The disruption of galactose metabolism is referred to as galactosemia. Classic galactosemia is an inherited deficiency in galactose 1-phosphate uridyl transferase activity. Symptoms: - vomiting, diarrhea after consuming milk, - enlargement of the liver, jaundice, sometimes cirrhosis, - cataracts, - lethargy and retarded mental development, - markedly elevated blood-galactose level - galactose is found in the urine. The absence of the transferase in red blood cells is a definitive diagnostic criterion. The most common treatment is to remove galactose (and lactose) from the diet.

Regulation of Glycolysis The rate glycolysis is regulated to meet two major cellular needs: (1) the production of ATP, and (2) the provision of building blocks for synthetic reactions. There are three control sites in glycolysis - the reactions catalyzed by hexokinase, phosphofructokinase 1, and pyruvate kinase These reactions are irreversible. Their activities are regulated by the reversible binding of allosteric effectors by covalent modification by the regulation of transcription (change of the enzymes amounts). The time required for allosteric control, regulation by phosphorylation, and transcriptional control is typically in milliseconds, seconds, and hours, respectively.

Phosphofructokinase 1 Is the Key Enzyme in the Control of Glycolysis Phosphofructokinase 1 is the most important control element in the mammalian glycolytic pathway. Phosphofructokinase 1 in the liver is a tetramer of four identical subunits. The positions of the catalytic and allosteric sites are identical.

High levels of ATP allosterically inhibit the phosphofructokinase 1 in the liver lowering its affinity for fructose 6-phosphate. AMP reverses the inhibitory action of ATP, and so the activity of the enzyme increases when the ATP/AMP ratio is lowered (glycolysis is stimulated as the energy charge falls). A fall in pH also inhibits phosphofructokinase 1 activity. The inhibition of phosphofructokinase by H+ prevents excessive formation of lactic acid and a precipitous drop in blood pH (acidosis). Phosphofructokinase 1 is inhibited by citrate, an early intermediate in the citric acid cycle. A high level of citrate means that biosynthetic precursors are abundant and additional glucose should not be degraded for this purpose.

Fructose 2,6-bisphosphate (F-2,6-BP) is a potent activator of phosphofructokinase 1. F-2,6-BP activates phosphofructokinase I by increasing its affinity for fructose 6-phosphate and diminishing the inhibitory effect of ATP. Fructose 2,6-bisphosphate is formed in a reaction catalyzed by phosphofructokinase 2 (PFK2), a different enzyme from phosphofructokinase 1. Fructose 2,6-bisphosphate is hydrolyzed to fructose 6-phosphate by a specific phosphatase, fructose bisphosphatase 2 (FBPase2). Both PFK2 and FBPase2 are present in a single polypeptide chain (bifunctional enzyme).

Regulation of Glycolysis by Fructose 2,6-bisphosphate When blood glucose level is low the glucagon is synthesized by pancreas Glucagon binds to cell receptors, stimulates the protein kinase A activity Protein kinase A phosphorylates the PFK-2 inhibiting its kinase activity and stimulating its phosphatase activity As result the amount of F-2,6-BP is decre-ased and glycolysis is slowed.

Regulation of Hexokinase Hexokinase is inhibited by its product, glucose 6-phosphate (G-6-P). High concentrations of G-6-P signal that the cell no longer requires glucose for energy, for glycogen, or as a source of biosynthetic precursors. Glucose 6-phosphate levels increase when glycolysis is inhibited at sites further along in the pathway. Glucose 6-phosphate inhibits hexokinase isozymes I, II and III. Glucokinase (isozyme IV) is not inhibited by glucose 6-phosphate. The role of glucokinase is to provide glucose 6-phosphate for the synthesis of glycogen.

Regulation of Pyruvate Kinase (PK) Several isozymic forms of pyruvate kinase are present in mammals (the L type predominates in liver, and the M type in muscle and brain). Fructose 1,6-bisphosphate allosterically activates pyruvate kinase. ATP allosterically inhibits pyruvate kinase to slow glycolysis when the energy charge is high. Finally, alanine (synthesized in one step from pyruvate) also allosterically inhibits the pyruvate kinases (signal that building blocks are abundant).

The isozymic forms of pyruvate kinase differ in their susceptibility to covalent modification. The catalytic properties of the L (liver) form—but not of the M (brain) form controlled by reversible phosphorylation. When the blood-glucose level is low, the glucagon leads to the phosphoryla-tion of pyruvate kinase, which diminishes its activity.

Regulation of Glycolysis Inhibition 1) PFK-1 is inhibited by ATP and citrate 2) Pyruvate kinase is inhibited by ATP and alanine 3) Hexokinase is inhibited by excess glucose 6-phosphate Stimulation 1) AMP and fructose 2,6-bisphosphate (F2,6BP) relieve the inhibition of PFK-1 by ATP 2) F1,6BP stimulate the activity of pyruvate kinase Alanine

Regulation of Hexose Transporters Several glucose transporters (GluT) mediate the thermodynamically downhill movement of glucose across the plasma membranes of animal cells. GluT is a family of 5 hexose transporters. Each member of this protein family consists of a single polypeptide chain forming 12 transmembrane segments. GLUT1 and GLUT3, present in erythrocytes, endothelial, neuronal and some others mammalian cells, are responsible for basal glucose uptake. Their Km value for glucose is about 1 mM. GLUT1 and GLUT3 continually transport glucose into cells at an essentially constant rate.

GLUT2, present in liver and pancreatic -cells has a very high Km value for glucose (15-20 mM). Glucose enters these tissues at a biologically significant rate only when there is much glucose in the blood. GLUT4, which has a Km value of 5 mM, transports glucose into muscle and fat cells. The presence of insulin leads to a rapid increase in the number of GLUT4 transporters in the plasma membrane. Insulin promotes the uptake of glucose by muscle and fat. The amount of this transporter present in muscle membranes increases in response to endurance exercise training. GLUT5, present in the small intestine, functions primarily as a fructose transporter.

The Pasteur Effect Under anaerobic conditions the conversion of glucose to pyruvate is much higher than under aerobic conditions (yeast cells produce more ethanol and muscle cells accumulate lactate) The Pasteur Effect is the slowing of glycolysis in the presence of oxygen. More ATP is produced under aerobic conditions than under anaerobic conditions, therefore less glucose is consumed aerobically.