Chapter 15.5 and 16.4

Hormones coordinate metabolic relations between different tissues, often by regulating the reversible modification of key enzymes. For instance, the hormone epinephrine triggers a signal-transduction cascade in muscle, resulting in the phosphorylation and activation of key enzymes and leading to the rapid deg-radation of glycogen to glucose, which is then used to supply ATP for muscle contraction.

Glucagon has the same effect in liver, but the glucose is released into the blood for other tissues to use. As described in Chapter 13, many hormones act through intracellular messengers, such as cyclic AMP and calcium ion, which coordinate the activities of many target proteins.

Phosphofructokinase Although the regulation of phosphofructokinase with respect to ATP is the same in the liver as in muscle, it is not as important in liver, because the ATP levels in liver do not fluctuate as they do in skeletal muscle. Low pH is not a metabolic signal for the liver enzyme, because lactate is not normally produced in the liver. Indeed, as we will see, lactate is converted into glucose in the liver.

Glycolysis also furnishes carbon skeletons for biosyntheses, and so a signal indicating whether building blocks are abundant or scarce should also regulate phosphofructokinase. In the liver, phosphofructokinase is inhibited by CITRATE , an early intermediate in the citric acid cycle (Chapter 19). A high level of Citrate in the cytoplasm means that biosynthetic precursors are abundant, and so there is no need to degrade additional glucose for this purpose. In this way, citrate enhances the inhibitory effect of ATP on phosphofructokinase.

Glycolysis in Muscle Is Regulated by Feedback Inhibition to Meet the Need for ATP

Glycolysis in skeletal muscle provides ATP primarily to power contraction. Consequently, the primary control of muscle glycolysis is the energy charge of the cell—the ratio of ATP to AMP. Glycolysis is stimulated as the energy charge falls—a signal that the cell needs more ATP. Let us examine how each of the key regulatory enzymes responds to changes in the amounts of ATP and AMP present in the cell.

Catalytic Activity is Regulated

- The catalytic activity of enzymes is controlled in several ways. -Reversible allosteric control is especially important. For example, the first reaction in many biosynthetic pathways is allosterically inhibited by the ultimate product of the pathway, an example of feedback inhibition This type of control can be almost instantaneous. Another recurring mechanism is the activation and deactivation of enzymes by reversible covalent modification . Reversible modification is often the end point of the signal-transduction cascades discussed in Chapter 13. For example, glycogen phosphorylase, the enzyme catalyzing the breakdown of glycogen, a storage form of glucose, is activated by the phosphorylation of a particular serine residue when glucose is scarce.

1. GLUT1 and GLUT3

-GLUT1 and GLUT3, present in nearly all mammalian cells, are responsible for transporting glucose into the cell under normal conditions. Like enzymes, transporters have Km values, except that, for transporters, Km is the concentration of the chemical transported that yields one-half maximal transport velocity. - The K m value for glucose for GLUT1 and GLUT3 is about 1 mM: ---------significantly less than the normal serum-glucose level, which typically ranges from 4 mM to 8 mM. Hence, GLUT1 and GLUT3 continuously transport glucose into cells at an essentially constant rate.

The Accessibility of Substrates Is Regulated

-In eukaryotes, metabolic regulation and flexibility are enhanced by compartmentalization. For example, fatty acid oxidation takes place in mitochondria, whereas fatty acid synthesis takes place in the cytoplasm. Compartmentalization segregates opposed reactions .

Hexokinase

-In the liver as well as in muscle, hexokinase is an important regulatory molecule. -The hexokinase reaction is controlled in the liver as in muscle. However, the enzyme primarily responsible for phosphorylating glucose in the liver is not hexokinase, but Glucokinase (hexokinase IV), an isozyme of hexokinase. -----Isozymes, or isoenzymes , are enzymes encoded by different genes with different amino acid sequences, yet they catalyze the same reaction. ------Isozymes usually differ in kinetic or regulatory properties.

Another means by which glycolysis in the liver responds to changes in blood glucose is through: ** the signal molecule** Fructose 2,6-bisphosphate (F-2,6-BP)= a potent Activator of phosphofructokinase. - After a meal rich in carbohydrates, the level of glucose in the blood rises (p. 293).

-In the liver, the concentration of fructose 6-phosphate rises when blood-glucose concentration is high because of the action of Hexokinase and Phosphoglucose isomerase, and the abundance of fructose 6-phosphate accelerates the synthesis of F-2,6-BP ( Figure 16.14 ). - Hence, an abundance of fructose 6-phosphate leads to a higher concentration of F-2,6-BP .

-Glucokinase phosphorylates glucose only when glucose is abundant, as would be the case after a meal. The reason is that glucokinase's affinity for glucose is about 50-fold lower than that of hexokinase, which means that glucokinase binds only to the glucose molecules that are in excess of what hexokinase can bind. -Further-more, glucokinase is not inhibited by its product, glucose 6-phosphate, as hexokinase is. -The role of glucokinase is to provide glucose 6-phosphate for the synthesis of glycogen and for the formation of fatty acids.

-The low affinity of glucokinase for glucose in the liver gives the brain and muscles first call for glucose when its supply is limited, and it ensures that glucose will not be wasted when it is abundant. Efforts are underway to develop drugs that will stimulate the activity of glucokinase as a treatment for excessive blood glucose seen in type 2 diabetes.

2. GLUT2

-present in liver and pancreatic Beta cells, is distinctive in having a very high Km value for glucose (15-20 mM). -Hence, glucose enters these tissues at a biologically significant rate only when there is much glucose in the blood. - The pancreas can thereby sense the glucose level and adjust the rate of insulin secretion accordingly. -Insulin signals the need to remove glucose from the blood for storage as glycogen or conversion into fat (Chapters 25 and 28). -The high Km value of GLUT2 also ensures that glucose rapidly enters liver cells only in times of plenty.

4. GLUT5

-present in the small intestine, functions primarily as a fructose transporter.

3. GLUT4

-which has a Km value of 5 mM, transports glucose into muscle and fat cells. -The number of GLUT4 transporters in the plasma membrane increases rapidly in the presence of insulin, which signals the presence of glucose in the blood. -Hence, insulin promotes the uptake of glucose by muscle and fat. Endurance exercise training increases the amount of GLUT4 present in muscle membranes.

Phosphofructokinase ---- Is the most-important control site in the mammalian glycolytic pathway High levels of ATP allosterically in-hibit the enzyme (a 340-kd tetramer). ATP binds to a specific regulatory site that is distinct from the catalytic site. The binding of ATP lowers the enzyme's affinity for fructose 6-phosphate. AMP reverses the inhibitory action of ATP. AMP competes with ATP for the binding site but, when bound, does not in-hibit the enzyme. Consequently the activity of the enzyme increases when the ATP/AMP ratio is lowered ( Figure 16.12 ).

A decrease in pH also inhibits phosphofructokinase activity by augmenting the inhibitory effect of ATP. The pH might fall when muscle is functioning anaerobically, producing excessive quantities of lactic acid. The inhibition of glycolysis, and therefore of lactic acid fermentation, protects the muscle from damage that would result from the accumulation of too much acid.

Metabolic Processes Are Regulated in Three Principle Ways

As is evident, the complex network of metabolic reactions must be rigorously regulated. At the same time, metabolic control must be flexible enough to adjust metabolic activity to the constantly changing external environments of cells. -Metabolism is regulated through the control of (1) the amounts of enzymes : Gene Expression, more need for these enzymes (2) their catalytic activities **Regulated by Allosteric (other sites; increasing or decreasing its affinity for its substrates) mechanisms R vs T state** (3) the accessibility of substrates

Figure 16.16 The control of the catalytic activity of pyruvate kinase.

Pyruvate kinase is regulated by allosteric effectors and covalent modification.

A Family of Transporters Enables Glucose to Enter and Leave Animal Cells

Several glucose transporters mediate the thermodynamically downhill move-ment of glucose across the plasma membranes of animal cells. Each member of this protein family, named GLUT1 to GLUT5, consists of a single polypeptide chain about 500 residues long ( Table 16.3 ).

Pyruvate kinase

Several isozymic forms of pyruvate kinase (a tetramer of 57-kd subunits) encoded by different genes are present in mammals: ----the L form predominates in liver, ----the M form in muscle and brain. -The L and M forms of pyruvate kinase have many properties in common. - Indeed, the liver enzyme behaves much as the muscle enzyme does in regard to allosteric regulation. -****However, the isozymic forms differ in their susceptibility to covalent modification, such as phosphorylation. ---The catalytic properties of the L form—but not of the M form—are also controlled by reversible phosphorylation ( Figure 16.16)

The Amounts of Enzymes Are Controlled

The amount of a particular enzyme depends on both its rate of synthesis and its rate of degradation. The level of many enzymes is adjusted primarily by a change in the rate of transcription of the genes encoding them (Section 15). -In E. coli , for example, the presence of lactose induces within minutes a more than 50-fold increase in the rate of synthesis of b -galactosidase, an enzyme required for the breakdown of this disaccharide.

16.4 The Glycolytic Pathway is tightly Controlled

The glycolytic pathway has a dual role: it degrades glucose to generate ATP and it provides building blocks for synthetic reactions, such as the formation of fatty acids and amino acids. The rate of conversion of glucose into pyruvate is regulated to meet these two major cellular needs. In metabolic pathways, enzymes catalyzing essentially irreversible reactions are potential sites of control. In glycolysis, the reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase are virtually irreversible, displaced far from equilibrium; hence, these enzymes would be expected to have regulatory as well as catalytic roles. In fact, each of them serves as a control site. These enzymes become more active or less so in response to the reversible binding of allosteric effec-tors or covalent modification. We will consider the control of glycolysis in two different tissues—skeletal muscle and liver

The Regulation of Glycolysis in the Liver Corresponds to the Biochemical Versatility of the Liver

The liver has a greater diversity of biochemical functions than muscle. Signifi-cantly, the liver maintains blood-glucose levels: it stores glucose as glycogen when glucose is plentiful, and it releases glucose when supplies are low. It also uses glu-cose to generate reducing power for biosynthesis (Chapter 26) as well as to synthesize a host of building blocks for other biomolecules. So, although the liver has many of the regulatory features of muscle glycolysis, the regulation of glycolysis in the liver is more complex.

Many reactions in metabolism are controlled by the energy status of the cell. One index of the energy status is the energy charge , which is the fraction of all of the adenine nucleotide molecules in the form of ATP plus half the fraction of adenine nucleotides in the form of ADP, given that ATP contains two phos-phoanhydride linkages, whereas ADP contains one. Hence, the energy charge is defined as:

The phosphorylation potential, in contrast with the energy charge, depends on the concentration of inorganic orthophosphate (Pi ).

Hypoxia itself enhances tumor growth by activating a transcription factor, hypoxia-inducible transcription factor (HIF-1). HIF-1 increases the expression of most glycolytic enzymes and the glucose transporters GLUT1 and GLUT3 ( Table 16.4 ). These adaptations by the cancer cells enable a tumor to survive until blood vessels can grow. HIF-1 also increases the expression of signal mol-ecules, such as vascular endothelial growth factor (VEGF), that facilitate the growth of blood vessels that will provide nutrients to the cells ( Figure 16.18 ). Without new blood vessels, a tumor would cease to grow and either die or re-main harmlessly small. Efforts are underway to develop drugs that inhibit the growth of blood vessels in tumors.

What biochemical alterations facilitate the switch to aerobic glycolysis? Again, the answers are not complete, but several factors appear to play a role. Lactate enhances the activity of HIF-1, stimulating the synthesis of yet more glycolytic enzymes. Moreover, changes in gene expression of isozymic forms of two glycolytic enzymes may be crucial. Tumor cells express an isozyme of hexokinase that binds to mitochondria. There, the enzyme has ready access to any ATP generated by oxidative phosphorylation and is no longer susceptible to feedback inhibition by its product, glucose 6-phosphate. An embryonic isozyme of pyruvate kinase also is expressed; it facilitates uses of glycolytic intermediates for biosynthetic reactions and is sensitive to growth-factor regulation.

Cancer and Exercise Training Affect Glycolysis in a Similar Fashion Tumors have been known for decades to display enhanced rates of glucose up-take and glycolysis. Indeed, rapidly growing tumor cells will metabolize glucose to lactate even in the presence of oxygen, a process called aerobic glycolysis or the "Warburg effect," after Otto Warburg, the biochemist who first noted this characteristic of cancer cells in the 1920s. In fact, tumors with a high glucose uptake are particularly aggressive, and the cancer is likely to have a poor prognosis. A non-metabolizable glucose analog, 2- 18 F-2- d -deoxyglucose, detectable by a combination of positron emission tomography (PET) and computer-aided tomography (CAT), easily visualizes tumors ( Figure 16.17 ).

What selective advantage does aerobic glycolysis offer the tumor over the energetically more efficient oxidative phosphorylation? Research is being actively pursued to answer the question, but we can speculate on the benefits. -First, aerobic glycolysis generates lactic acid that is then secreted. -Acidification of the tumor environment has been shown to facilitate tumor invasion and inhibit the immune system from attacking the tumor -Second, the increased uptake of glucose and the formation of glucose 6-phosphate provide substrates for another metabolic pathway, the pentose phosphate pathway (Chapter 26), which generates biosynthetic reducing power. -Moreover, the pentose phosphate pathway, in cooperation with glycolysis, produces precursors for biomolecules necessary for growth, such as nucleotides. Finally, cancer cells grow more rapidly than the blood vessels that nourish them; thus, as solid tumors grow, the oxygen concentration in their en-vironment falls. In other words, they begin to experience hypoxia, a deficiency of oxygen. The use of aerobic glycolysis reduces the dependence of cell growth on oxygen.

Why does AMP but not ADP stimulate the activity of phosphofructokinase?

When ATP is being utilized rapidly, the enzyme adenylate kinase can form ATP from ADP by the following reaction:

Hexokinase Phosphofructokinase is the most important regulatory enzyme in glycolysis, but it is not the only one. Hexokinase, the enzyme catalyzing the first step of glycolysis, is inhibited by its product, glucose 6-phosphate. High concentrations of glucose 6-phosphate signal that the cell no longer requires glucose for energy, and so no more glucose needs to be broken down. The glucose will then be left in the blood. A rise in glucose 6-phosphate concentration is a means by which phosphofructokinase communicates with hexokinase. When phosphofructokinase is inactive, the concentration of fructose 6-phosphate rises. In turn, the level of glucose 6-phosphate rises because it is in equilibrium with fructose 6-phosphate. Hence, the inhibition of phosphofructokinase leads to the inhibition of hexokinase .

Why is phosphofructokinase rather than hexokinase the pacemaker of gly-colysis? The reason becomes evident on noting that glucose 6-phosphate is not solely a glycolytic intermediate. In muscle, for example, glucose 6-phosphate can also be converted into glycogen. The first irreversible reaction unique to the gly-colytic pathway, the committed step (p. 112), is the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate. Thus, phosphofructokinase as the primary control site in glycolysis is highly appropriate. In general, the enzyme catalyzing the committed step in a metabolic sequence is the most-important control element in the pathway.