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METABOLIC ENGINEERING (BTG 406)

REGULATION OF CARBOHYDRATE METABOLISM

Glycolysis

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The Glycolytic Pathway

Regulation of glycolysis

The rate at which the glycolytic pathway operates is controlled primarily by allosteric regulation of three enzymes: hexokinase, PFK-1, and pyruvate kinase. The reactions catalyzed by these enzymes are irreversible and can be switched on and off by allosteric effectors. In general, allosteric effectors are molecules whose cellular concentrations are sensitive indicators of a cell's metabolic state. Some allosteric effectors are product molecules. For example, hexokinase is inhibited by excess glucose-6-phosphate. Several energy-related molecules also act as allosteric effectors. For example, a high AMP concentration (an indicator of low energy production) activates PFK-1 and pyruvate kinase. In contrast, a high ATP concentration (an indicator that the cell's energy requirements are being met) inhibits both enzymes. Citrate and acetyl-CoA, which accumulate when ATP is in rich supply, inhibit PFK-1 and pyruvate kinase, respectively. Fructose-2,6-bisphosphate, produced via hormone-induced covalent modification of PFK-2, is an indicator of high levels of available glucose and allosterically activates PFK-1.

Accumulated fructose- 1,6-bisphosphate activates pyruvate kinase, providing a feed-forward mechanism of control (i.e., fructose-1,6-bisphosphate is an allosteric activator).

Allosteric Regulation of Glycolysis

Enzyme Activator Inhibitor

Hexokinase Glucose-6-phosphate, ATP

PFK-1 Fructose-2,6-bisphosphate, AMP Citrate, ATP

Pyruvate kinase Fructose-l,6-bisphosphate, AMP Acetyl-CoA, ATP

Glucagon, present when serum glucose is low, activates the phosphatase function of PFK-2, reducing the level of fructose-2,6-bisphosphate in the cell. Insulin, present when serum glucose is high, activates the kinase function of PFK-2, increasing the level of fructose-2,6-bisphosphate in the cell.

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Regulation of fructose-2,6-bisphosphate

Citric acid cycle regulation

The citric acid cycle is precisely regulated so that the cell's energy and biosynthetic requirements are constantly met. Regulation is achieved primarily by the modulation of key enzymes and the availability of certain substrates. Because of its prominent role in energy production, the cycle also depends on a continuous supply of NAD+, FAD, and ADP.

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The Citric Acid Cycle

The citric acid cycle enzymes citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase are closely regulated because they catalyze reactions that represent important metabolic branch points.

1. Citrate synthase, the first enzyme in the cycle, catalyzes the formation of citrate from acetyl-CoA and oxaloacetate. Because the concentrations of acetyl-CoA and oxaloacetate are low in mitochondria in relation to the amount of the enzyme, any increase in substrate availability stimulates citrate synthesis. (Under these conditions the reaction is first order with respect to substrate. Therefore the rate of citrate synthesis is influenced by changes in concentrations of acetyl-CoA and oxaloacetate.) High concentrations of succinyl-CoA (a "downstream" intermediate product of the cycle) and citrate inhibit citrate synthase by acting as allosteric inhibitors. Other allosteric regulators of this reaction are NADH and ATP, whose concentrations reflect the cell's current energy status. A resting cell has high NADH/NAD+ and ATP/ADP ratios. As a cell becomes metabolically active, NADH and ATP concentrations decrease. Consequently, key enzymes such as citrate synthase become more active.

2. Isocitrate dehydrogenase catalyzes the second closely regulated reaction in the cycle. Its activity is stimulated by relatively high concentrations of ADP and NAD+ and inhibited by ATP and NADH. Isocitrate dehydrogenase is closely regulated because of its important role in citrate metabolism. The conversion of citrate to isocitrate is reversible. An equilibrium mixture of the two molecules consists largely of citrate. (The reaction is driven forward because isocitrate is rapidly transformed to α-ketoglutarate.) Of the two molecules, only citrate can penetrate the mitochondrial inner membrane. (When a substantial number of citrate molecules move into cytoplasm, the cell's current requirement for energy is low.) Citrate transport is used to transfer acetyl-CoA out of mitochondria because acetyl-CoA cannot penetrate the inner mitochondrial membrane. Once in the cytoplasm, citrate is cleaved by citrate lyase. The acetyl-CoA formed is used in several biosynthetic processes, such as fatty acid synthesis. Oxaloacetate is used in biosynthetic reactions, or it can be converted to malate. Malate either reenters the mitochondrion, where it is reconverted to oxaloacetate, or is converted in the cytoplasm to pyruvate

by malic enzyme. Pyruvate then reenters the mitochondrion. In addition to being a precursor of acetyl-CoA and oxaloacetate in the cytoplasm, citrate also acts directly to regulate several cytoplasmic processes. Citrate is an allosteric activator of the first reaction of fatty acid synthesis. In addition, citrate metabolism provides some of the NADPH used in fatty acid synthesis. Finally, because citrate is an inhibitor of PFK-1, it inhibits glycolysis.

3. The activity of α-ketoglutarate dehydrogenase is strictly regulated because of the important role of α-ketoglutarate in several metabolic processes (e.g., amino acid metabolism). When a cell's energy stores are low, α-ketoglutarate dehydrogenase is activated and α-ketoglutarate is retained within the cycle at the expense of biosynthetic processes. As the cell's supply of NADH rises, the enzyme is inhibited, and α-ketoglutarate molecules become available for biosynthetic reactions.

Two enzymes outside the citric acid cycle profoundly affect its regulation. The relative activities of pyruvate dehydrogenase and pyruvate carboxylase determine the degree to which pyruvate is used to generate energy and biosynthetic precursors. For example, if a cell is using a cycle intermediate such as α-ketoglutarate in biosynthesis, the concentration of oxaloacetate falls and acetyl-CoA accumulates. Because acetyl-CoA is an activator of pyruvate carboxylase (and an inhibitor of pyruvate dehydrogenase), more oxaloacetate is produced from pyruvate, thus replenishing the cycle.

Regulation of gluconeogenesis

The rate of gluconeogenesis is affected primarily by substrate availability, allosteric effectors, and hormones. Gluconeogenesis is stimulated by high concentrations of lactate, glycerol, and amino acids. A high-fat diet, starvation, and prolonged fasting make large quantities of these molecules available.

The four key enzymes in gluconeogenesis (pyruvate carboxylase, PEP carboxykinase, fructose-1, 6-bisphosphatase, and glucose-6-phosphatase) are affected to varying degrees by allosteric modulators. For example, fructose-1,6-bisphosphatase is activated by ATP and inhibited by AMP and fructose-2,6-bisphosphate.

Acetyl-CoA activates pyruvate carboxylase. (The concentration of acetyl-CoA, a product of fatty acid degradation, is especially high during starvation.)

Hormones affect gluconeogenesis by altering the concentrations of allosteric effectors and the rate key enzymes are synthesized. Glucagon depresses the synthesis of fructose-2,6-bisphosphate, activating the phosphatase function of PFK-2. The lowered concentration of fructose-2,6-bisphosphate reduces activation of PFK-1 and releases the inhibition of fructose-1,6-bisphosphatase.

Another effect of glucagon binding to liver cells is the inactivation of the glycolytic enzyme pyruvate kinase. (Protein kinase C, an enzyme activated by cAMP, converts pyruvate kinase to its inactive phosphorylated conformation.) Hormones also influence gluconeogenesis by altering enzyme synthesis. For example, the synthesis of gluconeogenic enzymes is stimulated by cortisol (a steroid hormone produced in the cortex of the adrenal gland). (Cortisol facilitates the body's adaptation to stressful situations. Its actions affect carbohydrate, protein, and lipid metabolism.) Finally, insulin action leads to the synthesis of new molecules of glucokinase, PFK-1, and PFK-2. Glucagon action leads to the synthesis of new molecules of PEP carboxykinase, fructose-l,6-bisphosphatase, and glucose-6-phosphatase.

The key point to remember is that it is the insulin/glucagon ratio that exerts the major regulatory effects on carbohydrate metabolism. After a carbohydrate meal, the insulin/glucagon ratio is high and glycolysis in the liver predominates over gluconeogenesis. After a period of fasting or following a high-fat, low-carbohydrate meal, the insulin/glucagon ratio is low and gluconeogenesis in the liver predominates over glycolysis. The availability of ATP is the second important regulator in the reciprocal control of glycolysis and gluconeogenesis in that high levels of AMP, the low-energy hydrolysis product of ATP, increase the flux through glycolysis at the expense of gluconeogenesis and low levels of AMP increase the flux through gluconeogenesis at the expense of glycolysis.

Although control at the PFK- l/fructose-l,6-bisphosphatase cycle would appear to be sufficient for this pathway, control at the pyruvate kinase step is key because it permits the maximal retention of PEP, a molecule with a very high phosphate transfer potential.

Regulation of glycogenesis and glycogenolysis

Glycogenesis (synthesis of glycogen from glucose) and glycogenolysis (breakdown of glycogen to glucose) are controlled by the enzymes glycogen synthase and glycogen phosphorylase, respectively. Regulation of these enzymes is accomplished by three mechanisms, namely: allosteric regulation, hormonal regulation, and influence of calcium.

l. Allosteric regulation of glycogen metabolism: There are certain metabolites that allosterically regulate the activities of glycogen synthase and glycogen phosphorylase. Synthesis of glycogen is increased when substrate availability and energy levels are high. On the other hand, glycogen breakdown is enhanced when glucose concentration and energy levels are low. In a well-fed state, there is high level of glucose-6-phosphate, which allosterically activates glycogen synthase for increased synthesis of glycogen. On the other hand, glucose-6-phosphate and ATP allosterically inhibit glycogen phosphorylase, thereby decreasing the breakdown of glycogen. Free glucose in liver also acts as an allosteric inhibitor of glycogen phosphorylase.

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Allosteric regulation of glycogenolysis and glycogenesis [pic]

2. Hormonal regulation of glycogen metabolism: The hormones, through a complex series of reactions, bring about covalent modification, namely phosphorylation and dephosphorylation of enzyme proteins that control the synthesis or degradation of glycogen synthesis.

Cyclic adenosine monophosphate (cAMP) as second messenger for hormones: Hormones like epinephrine and norepinephrine, and glucagon (in liver) activate adenylate cyclase to increase the production of cAMP. The enzyme phosphodiesterase breaks down cAMP. The hormone insulin increases the phosphodiesterase activity in liver and lowers the cAMP levels.

Regulation of glycogen synthesis by cAMP: Glycogen synthase exists in two forms; glycogen synthase 'a'-which is not phosphorylated and most active, and glycogen synthase 'b' as phosphorylated inactive form. Glycogen synthase 'a' can be converted to 'b' form (inactive) by phosphorylation. The degree of phosphorylation is proportional to the inactive state of enzyme. The process of phosphorylation is catalyzed by a cAMP-dependent protein kinase. The protein kinase phosphorylates and inactivates glycogen synthase by converting 'a' form to 'b' form. The glycogen synthase 'b' can be converted back to synthase 'a' by protein phosphatase l.

The inhibition of glycogen synthesis is brought by epinephrine (also norepinephrine) and glucagon through cAMP by converting active glycogen synthase 'a' to inactive synthase 'b'.

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Hormonal regulation of glycogen synthesis (glycogenesis)

Regulation of glycogen degradation by cAMP: Epinephrine and glucagon bring about glycogenolysis by their action on glycogen phosphorylase through cAMP. Glycogen phosphorylase exists in two forms, an active 'a' form and inactive form 'b'. The cAMP (formed due to hormonal stimulus) activates cAMP dependent protein kinase. This active protein kinase phosphorylates inactive form of glycogen phosphorylase kinase to active form. The enzyme protein phosphatase removes phosphate and inactivates phosphorylase kinase. The active phosphorylase kinase phosphorylates inactive glycogen phosphorylase 'b' to active glycogen phosphorylase 'a' which degrades glycogen. The enzyme protein phosphatase 1 can dephosphorylate and convert active glycogen phosphorylase 'a' to inactive 'b' form.

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Hormonal regulation of glycogen degradation (glycogenolysis)

3. Effect of Ca2+ ions on glycogenolysis: When muscle contracts, Ca2+ ions are released from the sarcoplasmic reticulum. Ca2+ binds to calmodulin-calcium modulating protein and directly activates phosphorylase kinase without the involvement of cAMP-dependent protein kinase. The overall effect of hormones on glycogen metabolism is that an elevated glucagon or epinephrine level increases glycogen degradation, whereas an elevated insulin results in increased glycogen synthesis.

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