Atp Pc

COa Pyruvate

QQpP OOOP

POOOP POOOP

OOOp OOOP

OOOP OOOp

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Figure 5.3 Glycolysis and gluconeogenesis steps. Glycolysis is a 10-step pathway that converts glucose to pyruvate. In gluconeogenesis, glucose is generated from substrates (e.g., pyruvate) by reversal of some of the glycolytic reactions and through the use of separate reactions. For details, see Sec. 5.2.3.

hydroxyl on carbon 1 that is phosphorylated in step 3, using another molecule of ATP. Phosphofructokinase-1 (PFK1), which catalyzes step 3, is a key glycolytic enzyme. It is tightly regulated by allosteric factors and hormones and will be discussed in more detail in Sec. 5.2.4. Through step 3 of glycolysis, the cell actually loses usable energy because two molecules of ATP have been consumed to drive early reactions.

Fructose 1,6-bisphosphate is cleaved, yielding the two 3-carbon sugars dihydroxyacetone phosphate and glyceraldehyde 3-phosphate in step 4 (Fig. 5.3). For glycolysis, dihydroxyacetone phosphate is converted to glyceraldehyde 3-phosphate by a reversible reaction in step 5. In step 6, the two glyceraldehyde 3-phosphate molecules are oxidized to 1, 3-bis-phosphoglycerates when they donate two pairs of electrons to two NAD+ to form two NADH. If oxygen is available, the NAD+ needed to continue glycolysis will be regenerated when the electrons carried by NADH are passed across the mitochondrial membrane and enter the electron transport chain. However, in conditions of limiting oxygen, NAD must be regenerated through fermentation as will be discussed later.

In step 7, two ATP are produced when phosphates are transferred from two 1,3-bisphosphoglycerate molecules to two ADP (Fig. 5.3). The direct transfer of a phosphate to ADP from an organic substrate is called substrate-level phosphorylation. As will be discussed later, most ATP is produced by oxidative phosphorylation that is coupled to electron transport in the mitochondrion (see Sec. 5.3.3). The net result of step 7 is generation of two molecules of ATP and two molecules of 3-phosphoglycerate. Hence, glycolysis has paid back the two ATP molecules consumed in earlier reactions.

ATP is again generated at the end of a series of reactions that converts two 3-phosphoglycerate molecules to two pyruvates (steps 8 to 10). First, in step 8, the phosphate in 3-phosphoglycerate is moved to the second carbon to produce 2-phosphoglycerate. In step 9, water is removed from 2-phosphoglycerate to produce phosphoenolpyruvate, a molecule that contains a high-energy phosphate bond. The enzyme pyruvate kinase (PK) catalyses transfer of the phosphates from the two phosphoenolpyruvates to two ADP forming two ATP and two molecules of pyruvate (step 10).

The two ATP molecules produced in step 10 constitute the two net

ATP generated during glycolysis.

Step 10 is highly exergonic (AG°' = —7.5 kcal/mol) and in effect is irreversible in the cell. Accordingly, conversion of pyruvate back to phos-phoenolpyruvate (and then to glucose) in gluconeogenesis requires two reactions to circumvent the substantial energy drop (see Sec. 5.2.3). This final irreversible step is critical in glycolysis; thus, it is not surprising that PK activity, like PFK1 activity, is tightly controlled by allosteric regulators and by hormones.

5.2.2 Fermentation

As indicated above, step 6 in glycolysis requires NAD+ as an electron acceptor (Fig. 5.3). If oxygen is available in animals, NAD+ is continually regenerated from NADH as the latter passes electrons, at least indirectly, to the electron transport chain in the mitochondrion. In the absence of oxygen, NAD+ is regenerated when NADH passes electrons to pyruvate, forming lactate (Fig. 5.4). This process is called lactate fermentation. Muscles commonly use lactate fermentation when the demands of strenuous exercise exceed the oxygen supply. In such cases, lactate accumulates in the muscle and then passes to the liver via the blood. In the liver, the lactate can be converted back to glucose via glu-coneogenesis and either stored as glycogen or released into the blood. Fermentation is by no means limited to muscle cells, because many cells produce lactate under anaerobic conditions.

Plant and yeast cells also use fermentation under anaerobic conditions but in these cells, the electrons are not passed to pyruvate. Pyruvate is first decarboxylated to acetaldehyde, which is then reduced by electrons from NADH to form ethanol (Fig. 5.4). Because the process yields ethanol, it is called alcoholic fermentation. Many industries, including those that produce wine and beer, rely on alcoholic fermentation in yeast.

5.2.3 Gluconeogenesis

In keeping with its name, gluconeogenesis is simply the creation of new glucose. Synthesis of new glucose requires "reversing" the reactions in the glycolytic pathway (Fig. 5.3) and obtaining the substrates needed to create the new glucose. Lactate and pyruvate are common glucose substrates, but amino acids and other sugars such as glycerol are also important.

Figure 5.4 Fermentation. Under anaerobic conditions, the NAD+ required for glycolysis must be regenerated by fermentation. In lactate fermentation (e.g., in animals), NAD+ is regenerated when NADH transfers electrons to pyruvate to form lactate. In alcoholic fermentation (e.g., in yeast), pyruvate is decarboxy-lated to acetaldehyde and the latter molecule accepts electrons from NADH. Steps 6,7-10 refer to the 10-step glycolysis pathway as pictured in Fig. 5.3.

Figure 5.4 Fermentation. Under anaerobic conditions, the NAD+ required for glycolysis must be regenerated by fermentation. In lactate fermentation (e.g., in animals), NAD+ is regenerated when NADH transfers electrons to pyruvate to form lactate. In alcoholic fermentation (e.g., in yeast), pyruvate is decarboxy-lated to acetaldehyde and the latter molecule accepts electrons from NADH. Steps 6,7-10 refer to the 10-step glycolysis pathway as pictured in Fig. 5.3.

Not all reactions in glycolysis can simply be reversed using the same enzyme. Steps 2 and 4 to 9 of Fig. 5.3 are reversible, but steps 1, 3, and particularly 10 require alternate pathways. Step 10 is one of the most difficult to reverse, because it is highly exergonic. The forward reaction is catalyzed by PK whereas the reverse is a two-reaction sequence catalyzed by pyruvate carboxylase (PC) and phosphoenolpyruvate car-boxykinase (PCK) (Fig. 5.3). PC adds carbon dioxide to a 3-carbon pyruvate to form a 4-carbon oxaloacetate. The energy needed to drive the reaction is derived from ATP. PCK then decarboxylates oxaloacetate and adds a phosphate from guanosine triphosphate (GTP) to form phosphoenolpyruvate. In step 3, PFK1 catalyzes the forward reaction whereas the reverse reaction is controlled by fructose 1,6-bisphosphatase (FBPase, Fig. 5.3). In step 1, HK catalyzes the forward reaction but glucose 6-phosphatase (GPase) catalyzes the reverse reaction (Fig. 5.3). Skeletal muscle expresses little GPase activity; thus, this tissue is unable to dephosphorylate glucose and release it into the blood. Glucose that enters skeletal muscle must be stored as glycogen, used for energy, or released back to the blood as lactate. The enzymes controlling steps 1, 3, and 10 play key roles in glycolysis and gluconeogenesis, and thus are tightly regulated by allosteric factors and hormones.

5.2.4 Regulation of anaerobic respiration

PFK1 and the opposing FBPase are two of the most tightly regulated enzymes in glycolysis and gluconeogenesis. Because PFK1 is a key enzyme in increasing energy production, its activity is inhibited alloster-ically by ATP and citrate, factors that reflect an abundance of energy in the cell. Conversely, PFK1 activity is increased by ADP and AMP, which are derived from dephosphorylation of ATP and indicate low cellular energy. AMP inhibits FBPase, and thus inhibits gluconeogenesis while increasing glycolysis.

A major activator of PFK1 and inhibitor of FBPase is fructose 2,6-bisphosphate (F 2,6-P). Production of F 2,6-P in the cell is controlled by a single enzyme that can express kinase (phosphofructokinase-2, PFK2) or phosphatase (fructose 2,6-bisphosphatase, F2,6BPase) activity (Fig. 5.5). Which activity is expressed by this bifunctional enzyme depends on its phosphorylation state. Phosphorylation of the bifunctional enzyme by protein kinase A (PKA) or another kinase leads to expression of phosphatase activity and F 2,6-P is converted to fructose 6-phosphate (F 6-P). If the bifunctional enzyme is not phosphorylated or if an existing phosphate is removed, then the enzyme expresses kinase activity and converts F 6-P to F 2,6-P. Thus, the unphosphorylated enzyme or PFK2 favors glycolysis by increasing the production of F 2,6-P, a strong activator of PFK1 (Fig. 5.5).

Figure 5.5 Regulation of anaerobic respiration. Fructose 2,6-bis-phosphate (F 2,6-P) stimulates the activity of phosphofructoki-nase-1 (PFK1) and inhibits the activity of fructose 1,6-bisphos-phatase (FBPase), key enzymes that catalyze the reactions in step 3 of glycolysis. For details, see Sec. 5.2.4.

The phosphorylation status of the PFK2/F2,6BPase is regulated by hormones. Epinephrine from the adrenal medulla and glucagon from the pancreas bind to membrane receptors and increase intracellular levels of the second messenger cyclic AMP (cAMP). Cyclic AMP activates PKA as part of the "normal" adenylyl cylase-cAMP pathway (see Sec. 4.4.1). PKA phosphorylates the bifunctional enzyme, promoting its F 2,6BPase activity and lowering cellular F 2,6-P. The net result is that epinephrine and glucagon promote gluconeogenesis and the release of glucose into the blood. In contrast, insulin increases PFK2 activity, which promotes glycolysis through increased production of F 2,6-P and subsequent activation of PFK1.

In summary, PFK1 and the opposing FBPase (glycolysis vs. gluconeo-genesis) are regulated by cellular allosteric factors that reflect local energy needs and by hormones that signal "global" changes in an organism.

Other key enzymes in glycolysis and gluconeogenesis also are regulated by allosteric factors and hormones. HK is inhibited by glucose 6-phosphate, an example of negative feedback by the product of an enzymatic reaction (Fig. 5.3). PK is inhibited by ATP, acetyl-CoA, and phos-phorylation. The first two factors are indicators of abundant energy and result from increased PK activity. Phosphorylation of PK is increased by PKA, which in turn is stimulated by glucagon and epi-nephrine through increased cAMP. Thus, epinephrine and glucagon inhibit PK activity as well as PFK1 activity. PK activity is increased by upstream generation of fructose 1,6-bisphosphate; thus, a decrease in PFK1 activity inhibits the activity of PK.

Figure 5.5 Regulation of anaerobic respiration. Fructose 2,6-bis-phosphate (F 2,6-P) stimulates the activity of phosphofructoki-nase-1 (PFK1) and inhibits the activity of fructose 1,6-bisphos-phatase (FBPase), key enzymes that catalyze the reactions in step 3 of glycolysis. For details, see Sec. 5.2.4.

PC and PCK, which catalyze the two-step pathway that reverses the reaction catalyzed by PK (Fig. 5.3), also are regulated. Acetyl-CoA stimulates PC activity, and PCK expression is regulated by numerous hormones. Insulin decreases expression of PCK, while epinephrine, glucagon, cortisol, and other gluconeogenic hormones increase its expression. One should bear in mind that the gluconeogenic actions of hormones primarily are expressed in tissues, such as liver, that express GPase activity and are capable of releasing glucose into the blood. In tissues like skeletal muscle that do not express GPase activity, the "gluconeogenic" hormones do not inhibit glycolysis, because inhibition would decrease production of needed energy and prevent release of lactic acid that feeds gluconeogenesis in the liver.

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