Glycolysis and gluconeogenesis

Glycolysis means sugar (glyco) breaking (lysis). It is an enzymatic pathway which converts glucose (a hexose, six carbon sugar) to two molecules of pyruvate (a triose, 3-carbon sugar). Glycolysis occurs in the cytoplasm and does not require the presence of oxygen. It is found (with variations in the terminal steps), in nearly all organisms and is one of the most ancient known metabolic pathways[1]. In aerobic organisms the pyruvate is used to generate more ATP via the citric acid cycle/cytochrome system, or converted into fatty acids and stored as triglycerides.

Gluconeogenesis is the reverse, a metabolic pathway that generates glucose from non-carbohydrate carbon substrates such as lactate, all citric acid cycle intermediates (through conversion to oxaloacetate), amino acids other than lysine or leucine, and glycerol. Transamination or deamination of amino acids allows their carbon skeleton to enter the cycle directly (as pyruvate or oxaloacetate), or indirectly via the citric acid cycle. Gluconeogenesis (with glycogenolysis) is one of the two main mechanisms which keep blood glucose levels from dropping too low (hypoglycemia). Gluconeogenesis takes place mainly in the liver and, to a lesser extent, in the cortex of kidneys. Gluconeogenesis occurs during fasting, low-carbohydrate intake or intense exercise, often in association with ketosis.

Enzymes involved in glycolysis

As shown in Figure 1, there are two phases and ten steps in the glycolytic pathway. The first five steps are regarded as the preparatory phase, since they consume energy to convert the glucose into two three-carbon sugar phosphates[1]. The first step is phosphorylation of glucose by hexokinase to form glucose-6-phosphate. This reaction consumes ATP, but acts to keep the glucose concentration low, promoting continuous transport of glucose into the cell through the plasma membrane transporters. In addition, it blocks glucose from leaking out as the cell lacks transporters for glucose-6-phosphate, and free diffusion out of the cell is prevented due to the charged nature of phosphorylated sugar. Liver also contains a glucokinase that has a much lower affinity for glucose (Km in the vicinity of normal glycemia), and differs in regulatory properties. The different substrate affinity and alternate regulation of this enzyme are a reflection of the role of the liver in maintaining blood sugar levels[1]. Glucose-6-phosphate is then converted into fructose-6-phosphate by the enzyme glucose phosphate isomerase. This reaction is freely reversible under normal cell conditions but is driven forward by the consumption of the product fructose-6-phosphate during the next step of glycolysis.

In glycolysis the enzymes involved in the preparatory phase are: 1, hexokinase / glucokinase; 2, glucose phosphate isomerase; 3, phosphofructokinase-1; 4, aldolase; 5, triosephosphate isomerase. The enzymes involved in the pay-off phase of glycolysis are: 6, glyceraldehydes-3-phosphate dehydrogenase; 7, phosphoglycerate kinase; 8, phosphoglycerate mutase; 9, enolase; 10, pyruvate kinase. Under anaerobic conditions pyruvate can be converted to lactate by lactate dehydrogenase, re-oxidising NADH to NAD and enabling glycolysis to continue to supply energy. The carbon sources for gluconeogensis can come from lactate, amino acids or glycerol, which enter the gluconeogenic pathway at different places. If lactate is the source then it is first converted to pyruvate by the enzyme lactate dehydrogenase. The enzymes involved in gluconeogensis from pyruvate are: I, pyruvate carboxylase in the mitochondria; II, phosphoenolpyruvate carboxykinase which converts oxaloacetate to phosphoenolpyruvate (occurs in both the mitochondria and the cytoplasm; mitochondial PEP can pass to the cytoplasm via special transport proteins; alternatively mitochondrial oxaloacetate can be reduced to malate which passes through the mitochondial membrane to the cytoplasm where the steps of gluconeogenesis continue); III, enolase; IV, phosphoglycerate mutase; V, phosphoglycerate kinase; VI, glyceraldehyde 3-phosphate dehydrogenase; VII, triosephosphate isomerase; VIII, aldolase; IX, fructose-1,6-bisphosphatase, the rate limiting step; X, glucose phosphate isomerase; XI, glucose-6-phosphatase.

The third step is the further phosphorylation of fructose-6-phosphate by phosphofructokinase 1 to produce fructose-1,6-bisphosphate. This second Figure 1. The enzyme steps involved in glycolysis (steps 1-11) and the reverse process gluconeogenesis (steps I – XI). Click for enlarged view.
Figure 1. The enzyme steps involved in glycolysis (steps 1-11) and the reverse process gluconeogenesis (steps I – XI). Click for enlarged view.
phosphorylation event is necessary to allow the formation of two charged groups (rather than only one) in the subsequent step of glycolysis, ensuring the prevention of free diffusion of substrates out of the cell.

Destabilizing the molecule in the previous reaction allows the hexose ring to be split by the enzyme aldolase (fourth step) into two triose sugars, dihydroxyacetone phosphate a ketone, and glyceraldehyde 3-phosphate an aldehyde that proceeds to the next step in the glycolytic pathway. The enzyme triosephosphate isomerase (fifth step) rapidly converts dihydroxyacetone phosphate to a second molecule of glyceraldehydes-3-phosphate that also proceeds further into glycolysis. This is advantageous, as it directs dihydroxyacetone phosphate down the same pathway as glyceraldehyde 3-phosphate, simplifying regulation.

The second half of glycolysis is known as the pay-off phase, characterised by a net gain of the energy-rich molecules ATP and NADH[1]. Since each glucose molecule leads to the formation of two triose sugars in the preparatory phase, each reaction in the pay-off phase occurs twice per glucose molecule (Figure 1).

In the sixth step, catalysed by the enzyme glyceraldehyde phosphate dehydrogenase, each triose sugar is dehydrogenated and inorganic phosphate added forming 1,3-bisphosphoglycerate and NADH. Phosphoglycerate kinase (seventh step) then transfers a phosphate group from the 1 position of 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. At this step, glycolysis has reached the break-even point: 2 molecules of ATP were consumed, and 2 new molecules have now been synthesized.

In the eighth step, the enzyme phosphoglycerate mutase converts 3-phosphoglycerate to 2-phosphoglycerate which in turn is converted to phosphoenolpyruvate by the enzyme enolase (ninth step). The final (tenth) step is the generation of pyruvate from phosphoenolpyruvate by the enzyme pyruvate kinase. This is the second substrate-level phosphorylation event which results in the production of another molecule of ATP per molecule of phosphoenolpyruvate.

Under anaerobic conditions such as muscle under severe exercise, the pyruvate can be converted to lactate by lactate dehydrogenase, re-oxidising NADH to NAD and enabling glycolysis to continue to supply energy. The lactate passes out into the blood stream and is absorbed by the liver for conversion into glucose and glycogen by gluconeogenesis and glycogenesis.

In summary, for every molecule of glucose degraded, the process consumes 2 molecules of ATP in the first portion of the pathway and generates 4 molecules of ATP in the second part of the process, giving a net yield of 2 ATP and 2 NADH molecules per molecule of glucose. Under aerobic conditions the reoxidation of the 2 NADH to 2 NAD+ via the cytochrome system in the mitochondria generates a further net gain of 4 ATP while under anaerobic conditions NADH is reoxidised by the conversion of pyruvate to lactate by lactate dehydrogenase. Glycolysis is thus an inefficient energy generating process compared to aerobic respiration where pyruvate is converted to acetyl CoA and oxidized to CO2 and water via the TCA cycle and cytochrome system. Here the 2 pyruvate molecules generated from each molecule of glucose generate a further 34 molecules of ATP[2].

Three dimensional structures of representatives of the enzymes involved in glycolysis have been determined[3].

Regulation of glycolysis

The rate limiting steps in the glycolytic pathway are: (i) the phosphorylation of glucose by hexokinase or glucokinase; (ii) the phosphorylation of fructose-6-phosphate to form fructose-1,6-bisphosphate by fructose-6-phosphate kinase; and (iii) the conversion of phosphoenolpyruvate to pyruvate by pyruvate kinase (Figure 1).

The generation of fructose-1,6-bisphosphate by phosphofructokinase-1 is a key regulatory point in the pathway and is also the rate-limiting step. The reaction is coupled to the hydrolysis of ATP and is, in essence, irreversible. Hence a different pathway must be used to do the reverse conversion during gluconeogensis. The most potent activator of phosphofructokinase-1 is fructose-2,6-bisphosphate, which is produced from fructose-6-phosphate by a second phosphofructokinase, phosphofructokinase-2. An abundance of fructose-6-phosphate results in a higher concentration of fructose-2,6-bisphosphate which on binding to phosphofructokinase-1 increases its affinity for fructose-6-phosphate and diminishes the inhibitory effect of ATP. This is an example of feed-forward stimulation as glycolysis is accelerated when glucose is abundant[4].

Phosphofructokinase-1 is allosterically inhibited by phosphoenolpyruvate and high levels of ATP while AMP reverses the inhibitory action of ATP. Therefore, the activity of the enzyme increases when the cellular ATP/AMP ratio is lowered. Glycolysis is thus stimulated when energy charge falls. Phosphofructokinase-1 has two sites with different affinities for ATP which is both a substrate for the kinase reaction and an allosteric inhibitor[4]. ATP binding to the allosteric site on phosphofructokinase 1 decreases the affinity of phosphofructokinase-1 for its substrate.

Phosphofructokinase-1 is also inhibited by low pH levels which augment the inhibitory effect of ATP. The pH falls when muscle is functioning anaerobically and producing excessive quantities of lactic acid. This inhibitory effect serves to protect the muscle from damage that would result from the accumulation of too much acid[5].

Phosphofructokinase-1 is inhibited indirectly by glucagon, whose binding to its receptor, activates adenyl cyclase generating cyclic AMP which activates protein kinase A. PKA phosphorylates phosphofructokinase-2, shutting off its kinase activity. This reverses any synthesis of fructose-2,6-bisphosphate from fructose-6-phosphate and thus inhibits phosphofructokinase-1[4].

Another key enzyme pyruvate kinase is regulated by its own substrate phosphoenolpyruvate and fructose-1,6-bisphosphate, the product generated in the third step of glycolysis (Figure X) [5]. Both enhance enzymatic activity. Thus, glycolysis is driven to operate faster when more substrate is present. ATP is a negative allosteric inhibitor providing parallel regulation (inhibition) with phosphofructokinase-1.

Liver pyruvate kinase is also regulated indirectly by epinephrine and glucagon through their ability to activate protein kinase A which phosphorylates liver pyruvate kinase to deactivate it. Muscle pyruvate kinase is not inhibited by epinephrine activation of protein kinase A. Thus, glycolysis is inhibited in the liver but unaffected in muscle when fasting. Insulin signaling activates phosphoprotein phosphatase-I, leading to dephosphorylation and activation of pyruvate kinase and the promotion of glycolysis[5].

Gluconeogenesis

Gluconeogenesis is a metabolic pathway that results in the generation of glucose from non-carbohydrate carbon substrates such as lactate, all citric acid cycle intermediates (through conversion to oxaloacetate), amino acids other than lysine or leucine, and glycerol. Transamination or deamination of amino acids facilitates the entering of their carbon skeleton into the cycle directly (as pyruvate or oxaloacetate), or indirectly via the citric acid cycle. Gluconeogenesis is one of the two main mechanisms humans and many other animals use to keep blood glucose levels from dropping too low (hypoglycemia). The other means of maintaining blood glucose levels is through the degradation of glycogen (glycogenolysis). Gluconeogenesis takes place mainly in the liver and, to a lesser extent, in the cortex of kidneys. This process occurs during periods of fasting, starvation, low-carbohydrate diets, or intense exercise and is often associated with ketosis. Gluconeogenesis is also a target of therapy for type II diabetes, such as metformin, which inhibits glucose formation and stimulates glucose uptake by cells[1].

Enzymes involved in gluconeogenesis

In all species, the formation of oxaloacetate from pyruvate and TCA cycle intermediates is restricted to the mitochondrion, whereas the enzymes that convert PEP to glucose are found in the cytosol. The location of the enzyme that links these two parts of gluconeogenesis by converting oxaloacetate to PEP, PEP carboxykinase, is variable by species: it can be found entirely within the mitochondria, entirely within the cytosol, or dispersed evenly between the two, as it is in humans. Transport of PEP across the mitochondrial membrane is accomplished by dedicated transport proteins; however no such proteins exist for oxaloacetate. Therefore in species that lack intra-mitochondrial PEP, oxaloacetate must be converted into malate or asparate, exported from the mitochondrion, and converted back into oxaloacetate in order to allow gluconeogenesis to continue[1].

As summarized in Figure 1, gluconeogenesis is a pathway consisting of eleven enzyme-catalyzed reactions. Many of the reactions are the reversible steps found in glycolysis. In the case where lactate is the source it is transported back to the liver where it is converted into pyruvate, the first designated substrate of the gluconeogenic pathway, by the enzyme lactate dehydrogenase. The pathway then begins in the mitochondria with the formation of oxaloacetate through carboxylation of pyruvate. This reaction also requires one molecule of ATP, and is catalyzed by pyruvate carboxylase. This enzyme is stimulated by high levels of acetyl-CoA (produced in β-oxidation of fatty acids in the liver) and inhibited by high levels of ADP. Mitochondial oxaloacetate is reduced to malate which passes through the mitochondial membrane to the cytoplasm where the steps of gluconeogenesis occur.

Oxaloacetate is decarboxylated and phosphorylated to produce phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. One molecule of GTP is hydrolyzed to GDP during this reaction. The next six steps in the pathway, catalysed by the enzymes enolase, phosphoglycerate mutase, phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase and aldolase (see Figure), are the same as reversed glycolysis. The next step where fructose-1,6-bisphosphatase converts fructose-1,6-bisphosphate to fructose 6-phosphate, is the rate-limiting step of gluconeogenesis. Fructose-6-phosphate is converted to glucose-6-phosphate by the glycolytic enzyme glucosephosphate isomerase. The final reaction of gluconeogenesis, the formation of glucose, occurs in the lumen of the endoplasmic reticulum, where glucose-6-phosphate is hydrolyzed by glucose-6-phosphatase to produce glucose. Glucose is shuttled into the cytosol by glucose transporters located in the membrane of the endoplasmic reticulum.

Regulation of Gluconeogenesis

While most steps in gluconeogenesis are the reverse of those found in glycolysis, three regulated and strongly exergonic reactions are replaced with more kinetically favorable reactions. Thus the glycolytic enzymes pyruvate kinase, phosphofructokinase, and hexokinase/glucokinase are replaced with PEP carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase. This system of reciprocal control where activation or inhibition of glycolysis is accompanied by the inhibition or activation of gluconeogenesis respectively avoids the formation of a futile cycle. The rate of gluconeogenesis is ultimately controlled by the action of the key enzyme, fructose-1,6-bisphosphatase, which is also regulated through signal transduction by cAMP and by phosphorylation. Most factors that regulate the activity of the gluconeogenesis pathway do so by inhibiting the activity or expression of key enzymes. However, both acetyl CoA and citrate activate gluconeogenesis enzymes (pyruvate carboxylase and fructose-1,6-bisphosphatase, respectively). Due to the reciprocal control of the cycle, acetyl-CoA and citrate also have inhibitory roles in the activity of pyruvate kinase[1].

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