(Photograph courtesy of Professor Melvin
18.3 ● Experimental Methods to Reveal Metabolic Pathways |
581 |
FIGURE 18.13 ● One of the earliest experiments using a radioactive isotope as a metabolic tracer. Cells of Chlorella (a green alga) synthesizing carbohydrate from carbon dioxide were exposed briefly (5 sec) to 14 C-labeled CO2. The products of CO2 incorporation were then quickly isolated from the cells, separated by two-dimensional paper chromatography, and observed via autoradiographic exposure of the chromatogram. Such experiments identified radioactive 3-phosphoglycerate (PGA) as the primary product of CO2 fixation. The 3-phos- phoglycerate was labeled in the 1-position (in its carboxyl group). Radioactive compounds arising from the conversion of 3-phosphoglycer- ate to other metabolic intermediates included phosphoenolpyruvate (PEP), malic acid, triose phosphate, alanine, and sugar phosphates and diphosphates.
Calvin, Lawmann Berkeley Laboratory, University of California, Berkeley.)
Heavy Isotopes
Heavy isotopes endow the compounds in which they appear with slightly greater masses than their unlabeled counterparts. These compounds can be separated and quantitated by mass spectrometry (or density gradient centrifugation, if they are macromolecules). For example, 18O was used in separate experiments as a tracer of the fate of the oxygen atoms in water and carbon dioxide to determine whether the atmospheric oxygen produced in photosynthesis arose from H2O, CO2, or both:
CO2 H2O 88n (CH2O) O2
If 18O-labeled CO2 was presented to a green plant carrying out photosynthesis, none of the 18O was found in O2. Curiously, it was recovered as H218O. In contrast, when plants fixing CO2 were equilibrated with H218O, 18O2 was evolved. These latter labeling experiments established that photosynthesis is best described by the equation
C16O2 2 H218O 88n (CH216O) 18O2 H216O
That is, in the process of photosynthesis, the two oxygen atoms in O2 come from two H2O molecules. One O is lost from CO2 and appears in H2O, and the other O of CO2 is retained in the carbohydrate product. Two of the four H atoms are accounted for in (CH2O), and two reduce the O lost from CO2 to H2O.
NMR as a Metabolic Probe
A technology analogous to isotopic tracers is provided by nuclear magnetic resonance (NMR) spectroscopy. The atomic nuclei of certain isotopes, such as the naturally occurring isotope of phosphorus, 31P, have magnetic moments. The resonance frequency of a magnetic moment is influenced by the local chemical environment. That is, the NMR signal of the nucleus is influenced in an identifiable way by the chemical nature of its neighboring atoms in the compound. In many ways, these nuclei are ideal tracers because their signals contain a great deal of structural information about the environment around the atom, and thus the nature of the compound containing the atom. Transformations of sub-
582 Chapter 18 ● Metabolism—An Overview
strates and metabolic intermediates labeled with magnetic nuclei can be traced by following changes in NMR spectra. Furthermore, NMR spectroscopy is a noninvasive procedure. Whole-body NMR spectrometers are being used today in hospitals to directly observe the metabolism (and clinical condition) of living subjects (Figure 18.14). NMR promises to be a revolutionary tool for clinical diagnosis and for the investigation of metabolism in situ (literally “in site,” meaning, in this case, “where and as it happens”).
Metabolic Pathways Are Compartmentalized Within Cells
Although the interior of a prokaryotic cell is not subdivided into compartments by internal membranes, the cell still shows some segregation of metabolism. For example, certain metabolic pathways, such as phospholipid synthesis and oxidative phosphorylation, are localized in the plasma membrane. Also, protein biosynthesis is carried out on ribosomes.
In contrast, eukaryotic cells are extensively compartmentalized by an endomembrane system. Each of these cells has a true nucleus bounded by a double membrane called the nuclear envelope. The nuclear envelope is continuous with the endomembrane system, which is composed of differentiated regions: the endoplasmic reticulum; the Golgi complex; various membranebounded vesicles such as lysosomes, vacuoles, and microbodies; and, ultimately, the plasma membrane itself. Eukaryotic cells also possess mitochondria and, if they are photosynthetic, chloroplasts. Disruption of the cell membrane and fractionation of the cell contents into the component organelles have allowed an analysis of their respective functions (Figure 18.15). Each compartment is dedicated to specialized metabolic functions, and the enzymes appropriate to these specialized functions are confined together within the organelle. In many instances, the enzymes of a metabolic sequence occur together within the organellar membrane. Thus, the flow of metabolic intermediates in the cell is spa-
(a)
|
Before |
|
exercise |
signalP |
Phosphocreatine |
|
31 |
|
|
|
|
|
Strength of |
|
|
ATP |
Pi |
γ |
β |
|
|
α |
10 |
0 |
–10 |
–20 ppm |
|
Chemical shift |
|
(b)
|
During |
|
exercise |
P signal |
Pi |
|
|
|
31 |
|
|
|
|
of |
|
|
|
|
Strength |
Phosphocreatine |
|
|
γ |
β |
|
α |
|
|
|
|
Chemical shift
FIGURE 18.14 ● With NMR spectroscopy one can observe the metabolism of a living subject in real time. These NMR spectra show the changes in ATP, creatine-P (phosphocreatine), and Pi levels in the forearm muscle of a human subjected to 19 minutes of exercise. Note that the three P atoms of ATP ( , , and ) have different chemical shifts, reflecting their different chemical environments.
18.3 ● Experimental Methods to Reveal Metabolic Pathways |
583 |
600rpm 
Tube is moved slowly up and down as pestle rotates.
Strain homogenate to remove connective
tissue and blood vessels.
Teflon pestle
Centrifuge homogenate at 600g × 10 min.
Tissue–sucrose homogenate (minced tissue + 0.25M sucrose buffer)
Supernatant 1 
Centrifuge supernatant 1 at 15,000g × 5 min.
Nuclei and any unbroken cells
Supernatant 2 
Centrifuge supernatant 2 at 100,000g × 60 min.
Mitochondria,
lysosomes,
and microbodies
Supernatant 3: 
Soluble fraction
of cytoplasm (cytosol)
Ribosomes and microsomes, consisting of endoplasmic reticulum, Golgi, and plasma membrane fragments
FIGURE 18.15 ● Fractionation of a cell extract by differential centrifugation. It is possible to separate organelles and subcellular particles in a centrifuge because their inherent size and density differences give them different rates of sedimentation in an applied centrifugal field. Nuclei are pelleted in relatively weak centrifugal fields, mitochondria in somewhat stronger fields, whereas very strong centrifugal fields are necessary to pellet ribosomes and fragments of the endomembrane system.
584 Chapter 18 ● Metabolism—An Overview
FIGURE 18.16 ● Compartmentalization of glycolysis, the citric acid cycle, and oxidative phosphorylation.
Glucose
|
Glucose |
|
|
ATP |
NADH |
|
Glycolysis |
|
ATP |
|
|
in the cytosol |
|
|
|
Acetyl– CoA |
|
NADH |
NADH |
|
Citric acid |
Citric |
cycle and |
ATP |
ATP |
oxidative |
|
|
acid |
phosphoryla- |
ATP |
ATP |
cycle |
tion in the |
|
mitochondria |
|
Pyruvate |
|
|
|
NADH |
NAD+ |
|
|
|
|
|
ADP |
ATP |
|
|
+ P |
|
|
|
H2O |
|
|
|
|
O2 |
CO2 |
tially as well as chemically segregated. For example, the 10 enzymes of glycolysis are found in the cytosol, but pyruvate, the product of glycolysis, is fed into the mitochondria. These organelles contain the citric acid cycle enzymes, which oxidize pyruvate to CO2. The great amount of energy released in the process is captured by the oxidative phosphorylation system of mitochondrial membranes and used to drive the formation of ATP (Figure 18.16).
18.4 ● Nutrition
The use of foods by organisms is termed nutrition. The ability of an organism to use a particular food material depends upon its chemical composition and upon the metabolic pathways available to the organism. In addition to essential fiber, food includes the macronutrients—protein, carbohydrate, and lipid—and the micronutrients—including vitamins and minerals.
Protein
Higher organisms must consume protein in order to make new proteins. Dietary protein is a rich source of nitrogen, and certain amino acids—the socalled essential amino acids—cannot be synthesized by higher organisms and can be obtained only in the diet. The average adult in the United States consumes far more protein than required for synthesis of essential proteins. Excess dietary protein is then merely a source of metabolic energy. Some of the amino acids (termed glucogenic) can be converted into glucose, whereas others, the ketogenic amino acids, can be converted to fatty acids and/or keto acids. If fat and carbohydrate are already adequate for the energy needs of the organism, then both kinds of amino acids will be converted to triacylglycerol and stored in adipose tissue.
A certain percentage of an organism’s own protein undergoes a constant process of degradation and resynthesis. Together with dietary protein, this recycled protein material participates in a nitrogen equilibrium or nitrogen balance. A positive nitrogen balance occurs whenever there is a net increase in the organism’s protein content, such as during periods of growth. A negative nitrogen balance exists when dietary intake of nitrogen is insufficient.
Carbohydrate
The principal purpose of carbohydrate in the diet is production of metabolic energy. Simple sugars are metabolized in the glycolytic pathway (see Chapter 19). Complex carbohydrates are degraded into simple sugars, which then can enter the glycolytic pathway. Carbohydrates are also essential components of nucleotides, nucleic acids, glycoproteins, and glycolipids. Human metabolism can adapt to a wide range of dietary carbohydrate levels, but the brain requires glucose for fuel. When dietary carbohydrate consumption exceeds the energy needs of the organism, excess carbohydrate is converted to triacylglycerols and glycogen for long-term energy storage. On the other hand, when dietary carbohydrate intake is low, ketone bodies are formed from acetate units to provide metabolic fuel for the brain.
Lipid
Fatty acids and triacylglycerols can be used as fuel by many tissues in the human body, and phospholipids are essential components of all biological membranes. Even though the human body can tolerate a wide range of fat intake levels, there are disadvantages in either extreme. Excess dietary fat is stored as triacylglycerols in adipose tissue, but high levels of dietary fat can also increase the risk of atherosclerosis and heart disease. Moreover, high dietary fat levels are also correlated with increased risk for colon, breast, and prostate cancers. When dietary fat consumption is low, there is a risk of essential fatty acid deficiencies. As seen in Chapter 25, the human body cannot synthesize linoleic and linolenic acids, so these must be acquired in the diet. Additionally, arachidonic
A D E E P E R L O O K
A Fad Diet—Low Carbohydrates, High Protein, High Fat
Possibly the most serious nutrition problem in the United States is excessive food consumption, and many people have experimented with fad diets in the hope of losing excess weight. One of the most popular of the fad diets has been the high-protein, high-fat (low-carbohydrate) diet. The premise for such diets is tantalizing: because the tricarboxylic acid (TCA) cycle (see Chapter 20) is the primary site of fat metabolism, and because glucose is usually needed to replenish intermediates in the TCA cycle, if carbohydrates are restricted in the diet, dietary fat should merely be converted to ketone bodies and excreted. This so-called diet appears to work at first because a low-carbohydrate diet results in an initial water (and weight) loss. This occurs because
glycogen reserves are depleted by the diet and because about 3 grams of water of hydration are lost for every gram of glycogen.
However, the long-term results of this diet are usually disappointing for several reasons. First, ketone body excretion by the human body usually does not exceed 20 grams (400 kJ) per day. Second, amino acids can function effectively to replenish TCA cycle intermediates, making the reduced carbohydrate regimen irrelevant. Third, the typical fare in a high-protein, high-fat, lowcarbohydrate diet is expensive but not very tasty, and it is thus difficult to maintain. Finally, a high-fat diet is a high risk factor for atherosclerosis and coronary artery disease.
586 Chapter 18 ● Metabolism—An Overview
acid can by synthesized in humans only from linoleic acid, so it too is classified as essential. The essential fatty acids are key components of biological membranes, and arachidonic acid is the precursor to prostaglandins, which mediate a variety of processes in the body.
Fiber
The components of food materials that cannot be broken down by human digestive enzymes are referred to as dietary fiber. There are several kinds of dietary fiber, each with its own chemical and biological properties. Cellulose and hemicellulose are insoluble fiber materials that stimulate regular function of the colon. They may play a role in reducing the risk of colon cancer. Lignins make up another class of insoluble fibers which absorb organic molecules in the digestive system. Lignins bind cholesterol and clear it from the digestive system, reducing the risk of heart disease. Pectins and gums are water-soluble fiber materials that form viscous gel-like suspensions in the digestive system, slowing the rate of absorption of many nutrients, including carbohydrates, and lowering serum cholesterol in many cases. The insoluble fibers are prevalent in vegetable grains. Water-soluble fiber is a component of fruits, legumes, and oats.
SPECIAL FOCUS:
VITAMINS
Vitamins are essential nutrients that are required in the diet, usually in trace amounts, because they cannot be synthesized by the organism itself. The requirement for any given vitamin depends on the organism. Not all “vitamins” are required by all organisms. Vitamins required in the human diet are listed in Table 18.4. These important substances are traditionally distinguished as being either water-soluble or fat-soluble. Except for vitamin C (ascorbic acid), the water-soluble vitamins are all components or precursors of important biological substances known as coenzymes. These are low-molecular-weight molecules that bring unique chemical functionality to certain enzyme reactions. Coenzymes may also act as carriers of specific functional groups, such as methyl groups and acyl groups. The side chains of the common amino acids provide only a limited range of chemical reactivities and carrier properties. Coenzymes, acting in concert with appropriate enzymes, provide a broader range of catalytic properties for the reactions of metabolism. Coenzymes are typically modified by these reactions and are then converted back to their original forms by other enzymes, so that small amounts of these substances can be used repeatedly. The coenzymes derived from the water-soluble vitamins are listed in Table 18.4. Each of these will be discussed in this chapter. The fat-soluble vitamins are not directly related to coenzymes, but they play essential roles in a variety of critical biological processes, including vision, maintenance of bone structure, and blood coagulation. The mechanisms of action of fat-soluble vitamins are not as well understood as their water-soluble counterparts, but modern research efforts are gradually closing this gap.
Vitamin B1: Thiamine and Thiamine Pyrophosphate
As shown in Figure 18.17, thiamine is composed of a substituted thiazole ring joined to a substituted pyrimidine by a methylene bridge. It is the precursor of thiamine pyrophosphate (TPP), a coenzyme involved in reactions of carbo-
FIGURE 18.17
Table 18.4
Vitamins and Coenzymes
Vitamin |
Coenzyme Form |
|
|
Water-Soluble |
|
Thiamine (vitamin B1) |
Thiamine pyrophosphate |
Niacin (nicotinic acid) |
Nicotinamide adenine dinucleotide (NAD ) |
|
Nicotinamide adenine dinucleotide phosphate |
|
(NADP ) |
Riboflavin (vitamin B2) |
Flavin adenine dinucleotide (FAD) |
|
Flavin mononucleotide (FMN) |
Pantothenic acid |
Coenzyme A |
Pyridoxal, pyridoxine, |
Pyridoxal phosphate |
pyridoxamine (vitamin B6) |
|
Cobalamin (vitamin B12) |
5 -Deoxyadenosylcobalamin |
|
Methylcobalamin |
Biotin |
Biotin-lysine complexes (biocytin) |
Lipoic acid |
Lipoyl-lysine complexes (lipoamide) |
Folic acid |
Tetrahydrofolate |
Fat-Soluble |
|
Retinol (vitamin A) |
|
Ergocalciferol (vitamin D2) |
|
Cholecalciferol (vitamin D3) |
|
-Tocopherol (vitamin E) |
|
Vitamin K |
|
|
|
hydrate metabolism in which bonds to carbonyl carbons (aldehydes or ketones) are synthesized or cleaved. In particular, the decarboxylations of -keto acids and the formation and cleavage of -hydroxyketones depend on thiamine pyrophosphate. The first of these is illustrated in Figure 18.18 by (a) the decarboxylation of pyruvate by yeast pyruvate decarboxylase to yield carbon dioxide and acetaldehyde. An example of the formation and cleavage of -hydroxyketones is presented in Figure 18.18 (b) the condensation of two molecules of pyruvate in the acetolactate synthase reaction. Another example is provided by a reaction from the pentose phosphate pathway (Chapters 22 and 23) called the transketolase reaction. This latter reaction is referred to as an -ketol transfer for obvious reasons.
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
O– |
O– |
|
|
H3C |
H |
H |
|
H3C |
H |
|
H |
|
|
|
|
|
|
|
|
|
|
|
O– |
|
|
C |
|
C |
|
OH |
|
C |
|
C |
|
|
O |
|
P |
|
O |
|
P |
|
|
NH2 |
H |
|
|
H |
H |
|
NH2 |
|
|
|
H |
|
H |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
TPP-synthetase |
|
H |
|
|
|
|
|
|
|
|
|
|
O |
O |
|
|
C |
|
N |
S |
|
|
+ ATP |
|
C |
|
N |
S |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
N |
|
H |
+ |
|
|
|
|
|
N |
H |
+ |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
H3C |
N |
|
|
|
H+ |
|
|
|
AMP |
H3C |
N |
|
H+ |
Acidic proton |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Thiamine (vitamin B1) |
|
|
|
|
|
|
|
Thiamine pyrophosphate (TPP) |
|
|
|
|
● Thiamine pyrophosphate (TPP), the active form of vitamin B1, is formed by the action of TPP-synthetase.
FIGURE 18.18
588 Chapter 18 ● |
Metabolism—An Overview |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
(a) |
|
|
|
O |
|
|
|
|
|
|
|
|
|
|
|
|
O |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Pyruvate |
|
|
|
|
|
|
|
|
H + |
|
|
|
|
|
|
|
|
|
|
An α |
-cleavage reaction |
CH3 |
|
C |
|
COO– |
|
CH3 |
|
|
C |
|
CO2 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
decarboxylase |
|
|
|
|
|
|
(b) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
O |
|
|
|
|
O |
|
|
|
|
|
|
|
|
|
|
|
O |
|
OH |
|
|
|
|
|
|
|
|
|
|
+ CH3 |
|
|
|
|
|
|
|
|
Acetolactate |
|
|
|
|
|
|
|
|
|
COO – + CO2 |
An α |
-condensation reaction |
CH3 |
|
C |
|
COO– |
|
|
C |
|
COO– |
|
CH3 |
|
C |
|
C |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
synthase |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
CH3
● Thiamine pyrophosphate participates in (a) the decarboxylation of-keto acids and (b) the formation and cleavage of -hydroxyketones.
Vitamins Containing Adenine Nucleotides
Several classes of vitamins are related to, or are precursors of, coenzymes that contain adenine nucleotides as part of their structure. These coenzymes include the flavin dinucleotides, the pyridine dinucleotides, and coenzyme A. The adenine nucleotide portion of these coenzymes does not participate actively in the reactions of these coenzymes; rather, it enables the proper enzymes to recognize the coenzyme. Specifically, the adenine nucleotide greatly increases both the affinity and the specificity of the coenzyme for its site on the enzyme, owing to its numerous sites for hydrogen bonding, and also the hydrophobic and ionic bonding possibilities it brings to the coenzyme structure.
Nicotinic Acid and the Nicotinamide Coenzymes
Nicotinamide is an essential part of two important coenzymes: nicotinamide adenine dinucleotide (NAD ) and nicotinamide adenine dinucleotide phosphate (NADP ) (Figure 18.19). The reduced forms of these coenzymes are NADH and NADPH. The nicotinamide coenzymes (also known as pyridine nucleotides) are electron carriers. They play vital roles in a variety of enzymecatalyzed oxidation–reduction reactions. (NAD is an electron acceptor in oxidative (catabolic) pathways and NADPH is an electron donor in reductive (biosynthetic) pathways.) These reactions involve direct transfer of hydride anion either to NAD(P) or from NAD(P)H. The enzymes that facilitate such
H U M A N B I O C H E M I S T R Y
Thiamine and Beriberi
Thiamine, whose structure is shown in Figure 18.17, is known as vitamin B1 and is essential for the prevention of beriberi, a nervous system disease that has occurred in the Far East for centuries and has resulted in considerable sickness and death in these countries. (As recently as 1958, it was the fourth leading cause of death in the Philippine Islands.) It was shown in 1882 by the directorgeneral of the medical department of the Japanese navy that beriberi could be prevented by dietary modifications. Ten years later, Christiaan Eijkman, a Dutch medical scientist working in Java, began research that eventually showed that thiamine was the
“anti-beriberi” substance. He found that chickens fed polished rice exhibited paralysis and head retractions and that these symptoms could be reversed if the rice polishings (the outer layers and embryo of the rice kernel) were fed to the birds. In 1911, Casimir Funk prepared a crystalline material from rice bran that cured beriberi in birds. He named it beriberi vitamine, because he viewed it as a “vital amine,” and thus he is credited with coining the word vitamin. The American biochemist R. R. Williams and his research group were the first to establish the structure of thiamine (in 1935) and a route for its synthesis.
FIGURE 18.19
|
|
|
|
|
|
|
|
Nicotinamide |
|
|
|
|
|
|
Nicotinamide |
|
|
|
|
|
|
|
|
(oxidized form) |
|
|
|
|
|
|
(reduced form) |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
pro-R pro-S |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
H |
O |
|
Hydride ion, |
|
|
|
|
O |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
H H |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
H |
– |
|
|
|
|
|
|
|
|
|
|
|
4 |
|
|
|
|
|
|
|
+ |
|
|
|
|
|
|
|
|
C NH2 |
|
|
|
|
|
|
|
C NH2 |
NAD |
|
|
|
|
|
|
|
5 |
|
3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
dinucleotide, |
|
|
|
|
|
|
|
6 |
N + |
2 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
... |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
N |
|
–O |
|
|
|
|
|
|
|
1 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
O |
|
CH2 |
O |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
adenine |
|
P |
|
|
|
H |
|
H |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
O |
|
|
|
|
|
|
NH2 |
|
|
|
|
|
|
|
|
|
|
O |
|
H |
OH OH |
H |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Nicotinamide |
|
O |
|
|
|
H |
|
H |
N |
|
|
|
N |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
P |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
–O |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
O |
|
CH |
2 |
O |
N |
N |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
AMP |
|
|
H |
|
|
|
H |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
OH OH |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
NADP+ contains a P on this 2'-hydroxyl
transfers are thus known as dehydrogenases. The hydride anion contains two electrons, and thus NAD and NADP act exclusively as two-electron carriers. The C-4 position of the pyridine ring, which can either accept or donate hydride ion, is the reactive center of both NAD and NADP. The quaternary nitrogen of the nicotinamide ring functions as an electron sink to facilitate hydride transfer to NAD , as shown in Figure 18.20. The adenine portion of the molecule is not directly involved in redox processes.
● The structures and redox states of the nicotinamide coenzymes. Hydride ion (H: , a proton with two electrons) transfers to NAD to produce NADH.
|
|
H |
B |
|
|
E |
H |
B |
|
E |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
O |
|
|
O |
|
|
|
|
|
|
... |
|
|
|
|
... |
|
|
|
|
... |
|
|
|
|
|
... |
|
C |
|
|
|
|
|
|
C |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
H |
|
|
|
|
|
|
|
|
|
|
|
H 
O
NH2
+N
R
Oxidized coenzyme (NAD+ or NADP+)
H H O
NH2
N
R
Reduced coenzyme (NADH or NADPH)
FIGURE 18.20 ● NAD and NADP participate exclusively in two-electron transfer reactions. For example, alcohols can be oxidized to ketones or aldehydes via hydride transfer to NAD(P) .
590 Chapter 18 ● Metabolism—An Overview
H U M A N B I O C H E M I S T R Y
Niacin and Pellegra
Pellegra, a disease characterized by dermatitis, diarrhea, and dementia, has been known for centuries. It was once prevalent in the southern part of the United States and is still a common problem in some parts of Spain, Italy, and Romania. Pellegra was once thought to be an infectious disease, but Joseph Goldberger showed early in this century that it could be cured by dietary actions. Soon thereafter, it was found that brewer’s yeast would prevent pellegra in humans. Studies of a similar disease in dogs, called blacktongue, eventually led to the identification of nicotinic acid as the relevant dietary factor. Elvehjem and his colleagues at the University of Wisconsin in 1937 isolated nicotinamide from liver, and showed that it and nicotinic acid could prevent and cure blacktongue in dogs. That same year, nicotin-
amide and nicotinic acid were both shown to be able to cure pellegra in humans. Interestingly, plants and many animals can synthesize nicotinic acid from tryptophan and other precursors, and nicotinic acid is thus not a true vitamin for these species. However, if dietary intake of tryptophan is low, nicotinic acid is required for optimal health. Nicotinic acid, which is beneficial to humans and animals, is structurally related to nicotine, a highly toxic tobacco alkaloid. In order to avoid confusion of nicotinic acid and nicotinamide with nicotine itself, niacin was adopted as a common name for nicotinic acid. Cowgill, at Yale University, suggested the name from the letters of three words—nicotinic, acid, and vitamin.
|
|
O |
|
|
|
|
COOH |
C |
NH2 |
|
N |
|
|
|
|
|
N |
N |
N |
|
N |
CH3 |
Pyridine |
Nicotinic acid |
Nicotinamide |
|
Nicotine |
The structures of pyridine, nicotinic acid, nicotinamide, and nicotine.
Examination of the structures of NADH and NADPH reveals that the 4- position of the nicotinamide ring is pro-chiral, meaning that while this carbon is not chiral, it would be if either of its hydrogens were replaced by something else. As shown in Figure 18.20, the hydrogen “projecting” out of the page toward you is the “pro-R” hydrogen because, if a deuterium is substituted at this position, the molecule would have the R-configuration. Substitution of the other hydrogen would yield an S -configuration. An interesting aspect of the enzymes that require nicotinamide coenzymes is that they are stereospecific and withdraw hydrogen from either the pro-R or the pro-S position selectively. This stereospecificity arises from the fact that enzymes (and the active sites of enzymes) are inherently asymmetric structures. These same enzymes are stereospecific with respect to the substrates as well.
The NADand NADP-dependent dehydrogenases catalyze at least six different types of reactions: simple hydride transfer, deamination of an amino acid to form an -keto acid, oxidation of -hydroxy acids followed by decarboxylation of the -keto acid intermediate, oxidation of aldehydes, reduction of isolated double bonds, and the oxidation of carbon–nitrogen bonds (as with dihydrofolate reductase).
Riboflavin and the Flavin Coenzymes
Riboflavin, or vitamin B2, is a constituent and precursor of both riboflavin 5 -phosphate, also known as flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The name riboflavin is a synthesis of the names for the molecule’s component parts, ribitol and flavin. The structures of riboflavin,
