Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

Biotin

Early in the 1900s, it was observed that certain strains of yeast required a material called bios for growth. Bios was eventually found to contain four different substances: myoinositol, -alanine, pantothenic acid, and a compound later shown to be biotin. Kögl and Tönnis first isolated biotin from egg yolk in 1936. Boas, in 1927, and Szent-György, in 1931, found substances in liver that were capable of curing and preventing the dermatitis, loss of hair, and paralysis that occurred in rats fed large amounts of raw egg whites (a condition known as egg white injury). Boas called the factor “protective factor X” and Szent-György named the substance vitamin H (from the German haut, meaning “skin”), but both were

soon shown to be identical to biotin. It is now known that egg white contains a basic protein called avidin, which has an extremely high affinity for biotin (K D 10 15 M). The sequestering of biotin by avidin is the cause of the egg white injury condition.

The structure of biotin was determined in the early 1940s by Kögl in Europe and by du Vigneaud and coworkers in the United States. Interestingly, the biotin molecule contains three asymmetric carbon atoms, and biotin could thus exist as eight different stereoisomers. Only one of these shows biological activity.

Lipoic Acid

Lipoic acid exists as a mixture of two structures: a closed-ring disulfide form and an open–chain reduced form (Figure 18.33). Oxidation–reduction cycles interconvert these two species. As is the case for biotin, lipoic acid does not often occur free in nature, but rather is covalently attached in amide linkage with lysine residues on enzymes. The enzyme that catalyzes the formation of the lipoamide linkage requires ATP and produces lipoamide-enzyme conjugates, AMP, and pyrophosphate as products of the reaction.

Lipoic acid is an acyl group carrier. It is found in pyruvate dehydrogenase and-ketoglutarate dehydrogenase, two multienzyme complexes involved in carbohydrate metabolism (Figure 18.34). Lipoic acid functions to couple acyl-group transfer and electron transfer during oxidation and decarboxylation of -keto acids.

The special properties of lipoic acid arise from the ring strain experienced by oxidized lipoic acid. The closed ring form is approximately 20 kJ higher in energy than the open-chain form, and this results in a strong negative reduction potential of about 0.30 V. The oxidized form readily oxidizes cyanides to isothiocyanates and sulfhydryl groups to mixed disulfides.

~1.5 nm

The biotin-lysine (biocytin) complex

● Biotin is covalently linked to a protein via the -amino group of a lysine residue. The biotin ring is thus tethered to the protein by a 10-atom chain. It functions by carrying carboxyl groups between distant sites on biotin-dependent enzymes.

Lipoic Acid

Lipoic acid (6,8-dithiooctanoic acid) was isolated and characterized in 1951 in studies that showed that it was required for the growth of certain bacteria and protozoa. This accomplishment was one of the most impressive feats of isolation in the early history of biochemistry. Eli Lilly and Co., in cooperation with Lester J. Reed at the University of Texas and I. C. Gunsalus at the

University of Illinois, isolated just 30 mg of lipoic acid from approximately 10 tons of liver! No evidence exists of a dietary lipoic acid requirement by humans; strictly speaking, it is not considered a vitamin. Nevertheless, it is an essential component of several enzymes of intermediary metabolism and is present in body tissues in small amounts.

Folic Acid, Pterins, and Insect Wings

Folic acid is a member of the vitamin B complex found in green plants, fresh fruit, yeast, and liver. Folic acid takes its name from folium, Latin for “leaf.” Pterin compounds are named from the Greek word for “wing” because these substances were first identified in insect wings. Two pterins are familiar to any child who has seen (and chased) the common yellow sulfur butterfly and its white counterpart, the cabbage butterfly. Xanthopterin and leu-

copterin are the respective pigments in these butterflies’ wings. Mammalian organisms cannot synthesize pterins; they derive folates from their diet or from microorganisms active in the intestines. Folic acid derives its name from folium, Latin for “leaf.” Pterin compounds are named from the Greek ´ , for “wing,” since these substances were first identified in insect wings.

dehydrogenase

FIGURE 18.34 ● The enzyme reactions catalyzed by lipoic acid.

may exist at the oxidation levels of methanol, formaldehyde, or formate (carbon atom oxidation states of –2, 0, and 2, respectively). The biosynthetic pathways for methionine and homocysteine (Chapter 26), purines (Chapter 27), and the pyrimidine thymine (Chapter 27) rely on the incorporation of onecarbon units from THF derivatives.

The Vitamin A Group

Vitamin A or retinol (Figure 18.36) often occurs in the form of esters, called retinyl esters. The aldehyde form is called retinal or retinaldehyde. Like all the fat-soluble vitamins, retinol is an isoprenoid molecule and is biosynthesized from isoprene building blocks (Chapter 8). Retinol can be absorbed in the diet from animal sources or synthesized from -carotene from plant sources. The absorption by the body of fat-soluble vitamins proceeds by mechanisms different from those of the water-soluble vitamins. Once ingested, preformed vitamin A or - carotene and its analogs are released from proteins by the action of proteolytic enzymes in the stomach and small intestine. The free carotenoids and retinyl esters aggregate in fatty globules that enter the duodenum. The detergent actions of bile salts break these globules down into small aggregates that can be digested by pancreatic lipase, cholesteryl ester hydrolase, retinyl ester hydrolase, and similar enzymes. The product compounds form mixed micelles (see Chapter 8) containing the retinol, carotenoids, and other lipids, which are absorbed into mucosal cells in the upper half of the intestinal tract. Retinol is esterified (usually with palmitic acid) and transported to the liver in a lipoprotein complex.

The retinol that is delivered to the retinas of the eyes in this manner is accumulated by rod and cone cells. In the rods (which are the better characterized of the two cell types), retinol is oxidized by a specific retinol dehydrogenase to become all-trans retinal and then converted to 11-cis retinal by reti-

Table 18.6

Oxidation States of Carbon in 1-Carbon Units Carried by Tetrahydrofolate

*Calculated by assigning valence bond electrons to the more electronegative atom and then counting the charge on the quasi ion. A carbon assigned four valence electrons would have an oxidation number of 0. The carbon in N 5-methyl-THF is assigned six electrons from the three COH bonds and thus has an oxidation number of 2.

† Note: All vacant bonds in the structures shown are to atoms more electronegative than C.

H2N N N H

N CH2 N R

O H

Folate

NADPH

+H+

NADP+

H

H2N N N

H

+H+

FIGURE 18.35 ● Formation of THF from folic acid by the dihydrofolate reductase reaction. The R group on these folate molecules symbolizes the one to seven (or more) glutamate units that folates characteristically contain. All of these glutamates are bound in-carboxyl amide linkages (as in the folic acid structure shown in the box A Deeper Look: Folic Acid, Pterins, and Insect Wings).The one-carbon units carried by THF are bound at N 5, or at

N 10, or as a single carbon attached to both N 5 and N 10.

-Carotene and Vision

Night blindness was probably the first disorder to be ascribed to a nutritional deficiency. The ancient Egyptians left records as early as 1500 B.C. of recommendations that the juice squeezed from cooked liver could cure night blindness if applied topically, and the method may have been known much earlier. Frederick Gowland Hopkins, working in England in the early 1900s, found that alcoholic extracts of milk contained a growth-stimulating fac-

tor. Marguerite Davis and Elmer McCollum at Wisconsin showed that egg yolk and butter contain a similar growth-stimulating lipid, which, in 1915, they called “fat soluble A.” Moore in England showed that -carotene, the plant pigment, could be converted to the colorless form of the liver-derived vitamin. In 1935, George Wald of Harvard showed that retinene found in visual pigments of the eye was identical with retinaldehyde, a derivative of vitamin A.

CH3 H3C

● (a) Vitamin D3 (cholecalciferol) is produced in the skin by the action of sunlight on 7-dehydrocholesterol. The successive action of mixed-function oxidases in the liver and kidney produces 1,25-dihydroxyvitamin D3, the active form of vitamin D.

(b) Ergocalciferol is produced in analogous fashion from ergosterol.

CH2

Vitamin D3 (cholecalciferol)

CH2

25-Hydroxyvitamin D3

CH3

CH3

CH3

Vitamin D and Rickets

Vitamin D is a family of closely related molecules that prevent rickets, a childhood disease characterized by inadequate intestinal absorption and kidney reabsorption of calcium and phosphate. These inadequacies eventually lead to the demineralization of bones. The symptoms of rickets include bowlegs,

knock-knees, curvature of the spine, and pelvic and thoracic deformities, the results of normal mechanical stresses on demineralized bones. Vitamin D deficiency in adults leads to a weakening of bones and cartilage known as osteomalacia.

Vitamin E

In a study of the effect of nutrition on reproduction in the rat in the 1920s, Herbert Evans and Katherine Bishop found that rats failed to reproduce on a diet of rancid lard, unless lettuce or whole wheat was added to the diet. The essential factor was traced to a vitamin in the wheat germ oil. Named vitamin E by Evans (using the next available letter following on the discovery of vita-

min D), the factor was purified by Emerson, who named it tocopherol, from the Greek tokos for “childbirth,” and pherein, for “to bring forth.” Vitamin E is now recognized as a generic term for a family of substances, all of them similar in structure to the most active form, -tocopherol.

CH3

Vitamin E (α -tocopherol)

FIGURE 18.38 ● The structure of vitamin E ( -tocopherol).

ergot, a rye fungus.) Because humans can produce vitamin D3 from 7–dehy- drocholesterol by the action of sunlight on the skin, “vitamin D” is not strictly speaking a vitamin at all.

On the basis of its mechanism of action in the body, cholecalciferol should be called a prohormone, a hormone precursor. Dietary forms of vitamin D are absorbed through the aid of bile salts in the small intestine. Whether absorbed in the intestine or photosynthesized in the skin, cholecalciferol is then transported to the liver by a specific vitamin D–binding protein (DBP), also known as transcalciferin. In the liver, cholecalciferol is hydroxylated at the C-25 position by a mixed-function oxidase to form 25-hydroxyvitamin D (that is, 25-hydrox- ycholecalciferol). Although this is the major circulating form of vitamin D in the body, 25-hydroxyvitamin D possesses far less biological activity than the final active form. To form this latter species, 25-hydroxyvitamin D is returned to the circulatory system and transported to the kidneys. There it is hydroxylated at the C-1 position by a mitochondrial mixed-function oxidase to form 1,25-dihy- droxyvitamin D3 (that is, 1,25-dihydroxycholecalciferol), the active form of vitamin D. 1,25-Dihydroxycholecalciferol is then transported to target tissues, where it acts like a hormone to regulate calcium and phosphate metabolism.

1,25-Dihydroxyvitamin D3, together with two peptide hormones, calcitonin and parathyroid hormone (PTH), functions to regulate calcium homeostasis and plays a role in phosphorus homeostasis. As described elsewhere in this text, calcium is important for many processes, including muscle contraction, nerve impulse transmission, blood clotting, and membrane structure. Phosphorus, of course, is of critical importance to DNA, RNA, lipids, and many metabolic processes. Phosphorylation of proteins is an important regulatory signal for many biological processes. Phosphorus and calcium are also critically important for the formation of bones. Any disturbance of normal serum phosphorus and calcium levels will result in alterations of bone structure, as in rickets. The mechanism of calcium homeostasis involves precise coordination of calcium (a) absorption in the intestine, (b) deposition in the bones, and (c) excretion by the kidneys. If a decrease in serum calcium occurs, vitamin D is converted to its active form, which acts in the intestine to increase calcium absorption. PTH and vitamin D act on bones to enhance absorption of calcium, and PTH acts on the kidney to cause increased calcium reabsorption. If serum calcium levels get too high, calcitonin induces calcium excretion from the kidneys and inhibits calcium mobilization from bone, while inhibiting vitamin D metabolism and PTH secretion.

Vitamin E: Tocopherol

The structure of vitamin E in its most active form, -tocopherol, is shown in Figure 18.38. -Tocopherol is a potent antioxidant, and its function in animals and humans is often ascribed to this property. On the other hand, the molecular details of its function are almost entirely unknown. One possible role for

O

Vitamin K2

(menaquinone series)

vitamin E may relate to the protection of unsaturated fatty acids in membranes because these fatty acids are particularly susceptible to oxidation. When human plasma levels of -tocopherol are low, red blood cells are increasingly subject to oxidative hemolysis. Infants, especially premature infants, are deficient in vitamin E. When low-birth-weight infants are exposed to high oxygen levels for the purpose of alleviating respiratory distress, the risk of oxygen-induced retina damage can be reduced with vitamin E administration. The mechanism(s) of action of vitamin E remain obscure.

Vitamin K: Naphthoquinone

The function of vitamin K (Figure 18.39) in the activation of blood clotting was not elucidated until the early 1970s, when it was found that animals and humans treated with coumarin-type anticoagulants contained an inactive form of prothrombin (an essential protein in the coagulation cascade). It was soon shown that a post-translational modification of prothrombin is essential to its function. In this modification, 10 glutamic acid residues on the amino terminal end of prothrombin are carboxylated to form -carboxyglutamyl residues. These residues are effective in the coordination of calcium, which is required for the coagulation process. The enzyme responsible for this modification, a liver microsomal glutamyl carboxylase, requires vitamin K for its activity (Figure 18.40). Not only prothrombin (called “factor II” in the clotting pathway) but also clotting factors VII, IX, and X and several plasma proteins—proteins C, M, S, and Z— contain -carboxyglutamyl residues in a manner similar to prothrombin. Other examples of -carboxyglutamyl residues in proteins are known.

CO2

Vitamin K–dependent glutamyl carboxylase

● The glutamyl carboxylase reaction is vitamin K–dependent. This enzyme activity is essential for the formation of -car- boxyglutamyl residues in several proteins of the blood-clotting cascade (Figure 15.5), accounting for the vitamin K dependence of coagulation.

Vitamin K and Blood Clotting

In studies in Denmark in the 1920s, Henrik Dam noticed that chicks fed a diet extracted with nonpolar solvents developed hemorrhages. Moreover, blood taken from such animals clotted slowly. Further studies by Dam led him to conclude in 1935 that the antihemorrhage factor was a new fat-soluble vitamin, which he called vitamin K (from koagulering, the Danish word for “coagulation”).

Dam, along with Karrar of Zurich, isolated the pure vitamin from alfalfa as a yellow oil. Another form, which was crystalline at room temperature, was soon isolated from fish meal. These two compounds were named vitamins K1 and K2. Vitamin K2 can actually occur as a family of structures with different chain lengths at the C-3 position.

608 Chapter 18 ● Metabolism—An Overview

PROBLEMS

1.If 3 1014 kg of CO2 are cycled through the biosphere annually, how many human equivalents (70-kg persons composed of 18% carbon by weight) could be produced each year from this amount of CO2?

2.Define the differences in carbon and energy metabolism between photoautotrophs and photoheterotrophs, and between chemoautotrophs and chemoheterotrophs.

3.Name three principal inorganic sources of oxygen atoms that are commonly available in the inanimate environment and readily accessible to the biosphere.

4.What are the features that generally distinguish pathways of catabolism from pathways of anabolism?

5.Name the three principal modes of enzyme organization in metabolic pathways.

6.Why do metabolic pathways have so many different steps?

7.Why is the pathway for the biosynthesis of a biomolecule at least partially different from the pathway for its catabolism? Why is the pathway for the biosynthesis of a biomolecule inherently more complex than the pathway for its degradation?

8.What are the metabolic roles of ATP, NAD , and NADPH?

9.Metabolic regulation is achieved via regulating enzyme activity in three prominent ways: allosteric regulation, covalent modi-

FURTHER READING

Atkinson, D. E., 1977. Cellular Energy Metabolism and Its Regulation. New York: Academic Press. A monograph on energy metabolism that is filled with novel insights regarding the ability of cells to generate energy in a carefully regulated fashion while contending with the thermodynamic realities of life.

Boyer, P. D., 1970. The Enzymes, 3rd ed. New York: Academic Press. A good reference source for the mechanisms of action of vitamins and coenzymes.

Boyer, P. D., 1970. The Enzymes, Vol. 6. New York: Academic Press. See discussion of carboxylation and decarboxylation involving TPP, PLP, lipoic acid, and biotin; B12-dependent mutases.

Boyer, P. D., 1972. The Enzymes, Vol. 7. New York: Academic Press. See especially elimination reactions involving PLP.

Boyer, P. D., 1974. The Enzymes, Vol. 10. New York: Academic Press. See discussion of pyridine nucleotide-dependent enzymes.

Boyer, P. D., 1976. The Enzymes, Vol. 13. New York: Academic Press. See discussion of flavin-dependent enzymes.

Cooper, T. G., 1977. The Tools of Biochemistry. New York: Wiley-Interscience. Chapter 3, “Radiochemistry,” discusses techniques for using radioisotopes in biochemistry.

fication, and enzyme synthesis and degradation. Which of these three modes of regulation is likely to be the quickest; which the slowest? For each of these general enzyme regulatory mechanisms, cite conditions in which cells might employ that mode in preference to either of the other two.

10.What are the advantages of compartmentalizing particular metabolic pathways within specific organelles?

11.Maple-syrup urine disease (MSUD) is an autosomal recessive genetic disease characterized by progressive neurological dysfunction and a sweet, burnt-sugar or maple-syrup smell in the urine. Affected individuals carry high levels of branched-chain amino acids (leucine, isoleucine, and valine) and their respective branched-chain -keto acids in cells and body fluids. The genetic defect has been traced to the mitochondrial branched-chain - keto acid dehydrogenase (BCKD). Affected individuals exhibit mutations in their BCKD, but these mutant enzymes exhibit normal levels of activity. Nonetheless, treatment of MSUD patients with substantial doses of thiamine can alleviate the symptoms of the disease. Suggest an explanation for the symptoms described and for the role of thiamine in ameliorating the symptoms of MSUD.

DeLuca, H., and Schnoes, H., 1983. Vitamin D: Recent advances. Annual Review of Biochemistry 52:411–439.

Jencks, W. P., 1969. Catalysis in Chemistry and Enzymology. New York: McGrawHill.

Knowles, J. R., 1989. The mechanism of biotin-dependent enzymes. Annual Review of Biochemistry 58:195–221.

Page, M. I., and Williams, A., eds., 1987. Enzyme Mechanisms. London: Royal Society of London.

Reed, L., 1974. Multienzyme complexes. Accounts of Chemical Research 7:40–46.

Srere, P. A., 1987. Complexes of sequential metabolic enzymes. Annual Review of Biochemistry 56:89–124. A review of how enzymes in some metabolic pathways are organized into complexes.

Walsh, C. T., 1979. Enzymatic Reaction Mechanisms. San Francisco: W. H. Freeman.

Chapter 19

Glycolysis

Louis Pasteur in his laboratory. Pasteur’s scientific investigations into fermentation of sugar were sponsored by the

French wine industry. (Albert Edelfelt, Musee d’Orsay, Paris;

Giraudon/Art Resource, New York)

Nearly every living cell carries out a catabolic process known as glycolysis— the stepwise degradation of glucose (and other simple sugars). Glycolysis is a paradigm of metabolic pathways. Carried out in the cytosol of cells, it is basically an anaerobic process; its principal steps occur with no requirement for oxygen. Living things first appeared in an environment lacking O2, and glycolysis was an early and important pathway for extracting energy from nutrient molecules. It played a central role in anaerobic metabolic processes during the first 2 billion years of biological evolution on earth. Modern organisms still employ glycolysis to provide precursor molecules for aerobic catabolic pathways (such as the tricarboxylic acid cycle) and as a short-term energy source when oxygen is limiting.

Living organisms, like machines, conform to the law of conservation of energy, and must pay for all their activities in the currency of catabolism.

ERNEST BALDWIN, Dynamic Aspects of Biochemistry (1952)

OUTLINE

glycolysis ● from the Greek glyk-, sweet, and lysis, splitting

609

610 Chapter 19 ● Glycolysis

Glycolysis

19.1 ● Overview of Glycolysis

An overview of the glycolytic pathway is presented in Figure 19.1. Most of the details of this pathway (the first metabolic pathway to be elucidated) were worked out in the first half of the 20th century by the German biochemists Otto Warburg, G. Embden, and O. Meyerhof. In fact, the sequence of reactions in Figure 19.1 is often referred to as the Embden–Meyerhof pathway.

Glycolysis consists of two phases. In the first, a series of five reactions, glucose is broken down to two molecules of glyceraldehyde-3-phosphate. In the second phase, five subsequent reactions convert these two molecules of glyc- eraldehyde-3-phosphate into two molecules of pyruvate. Phase 1 consumes two molecules of ATP (Figure 19.2). The later stages of glycolysis result in the production of four molecules of ATP. The net is 4 2 2 molecules of ATP produced per molecule of glucose.

Rates and Regulation of Glycolytic Reactions Vary Among Species

Microorganisms, plants, and animals (including humans) carry out the 10 reactions of glycolysis in more or less similar fashion, although the rates of the individual reactions and the means by which they are regulated differ from species to species. The most significant difference among species, however, is the way in which the product pyruvate is utilized. The three possible paths for pyruvate are shown in Figure 19.1. In aerobic organisms, including humans, pyruvate is oxidized (with loss of the carboxyl group as CO2), and the remaining two-carbon unit becomes the acetyl group of acetyl-coenzyme A. This acetyl group is metabolized by the tricarboxylic acid cycle (and fully oxidized) to yield CO2. The electrons removed in this oxidation process are subsequently passed through the mitochondrial electron transport system and used to generate molecules of ATP by oxidative phosphorylation, thus capturing most of the metabolic energy available in the original glucose molecule.

19.2 ● The Importance of Coupled Reactions in Glycolysis

The process of glycolysis converts some, but not all, of the metabolic energy of the glucose molecule into ATP. The free energy change for the conversion of glucose to two molecules of lactate (the anaerobic route shown in Figure 19.1) is 183.6 kJ/mol:

G 183.6 kJ/mol

This process occurs with no net oxidation or reduction. Although several individual steps in the pathway involve oxidation or reduction, these steps compensate each other exactly. Thus, the conversion of a molecule of glucose to two molecules of lactate involves simply a rearrangement of bonds, with no net loss or gain of electrons. The energy made available through this rearrangement into a more stable (lower energy) form is a relatively small part of the total energy obtainable from glucose.

The production of two molecules of ATP in glycolysis is an energy-requir- ing process:

G° 2 30.5 kJ/mol 61.0 kJ/mol