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

localized within different cellular compartments. Isolating opposing activities within distinct compartments, such as separate organelles, avoids interference between them. For example, the enzymes responsible for catabolism of fatty acids, the fatty acid oxidation pathway, are localized within mitochondria. In contrast, fatty acid biosynthesis takes place in the cytosol. In subsequent chapters, we shall see that the particular molecular interactions responsible for the regulation of metabolism become important to an understanding and appreciation of metabolic biochemistry.

Modes of Enzyme Organization in Metabolic Pathways

The individual metabolic pathways of anabolism and catabolism consist of sequential enzymatic steps (Figure 18.5). Several types of organization are possible. The enzymes of some multienzyme systems may exist as physically separate, soluble entities, with diffusing intermediates (Figure 18.5a). In other instances, the enzymes of a pathway are collected to form a discrete multienzyme complex, and the substrate is sequentially modified as it is passed along from enzyme to enzyme (Figure 18.5b). This type of organization has the advantage that intermediates are not lost or diluted by diffusion. In a third pattern of organization, the enzymes common to a pathway reside together as a membrane-bound system (Figure 18.5c). In this case, the enzyme participants (and perhaps the substrates as well) must diffuse in just the two dimensions of the membrane to interact with their neighbors.

As research reveals the ultrastructural organization of the cell in ever greater detail, more and more of the so-called soluble enzyme systems are found to be physically united into functional complexes. Thus, in many (perhaps all) metabolic pathways, the consecutively acting enzymes are associated into stable multienzyme complexes that are sometimes referred to as metabolons, a word meaning “units of metabolism.”

FIGURE 18.5 ● Schematic representation of types of multienzyme systems carrying out a metabolic pathway: (a) Physically separate, soluble enzymes with diffusing intermediates. (b) A multienzyme complex. Substrate enters the complex, becomes covalently bound and then sequentially modified by enzymes E1 to E5 before product is released. No intermediates are free to diffuse away. (c) A membranebound multienzyme system.

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The Pathways of Catabolism Converge to a Few End Products

If we survey the catabolism of the principal energy-yielding nutrients (carbohydrates, lipids, and proteins) in a typical heterotrophic cell, we see that the degradation of these substances involves a succession of enzymatic reactions. In the presence of oxygen (aerobic catabolism), these molecules are degraded ultimately to carbon dioxide, water, and ammonia. Aerobic catabolism consists of three distinct stages. In stage 1, the nutrient macromolecules are broken down into their respective building blocks. Given the great diversity of macromolecules, these building blocks represent a rather limited number of products. Proteins yield up their 20 component amino acids, polysaccharides give rise to carbohydrate units that are convertible to glucose, and lipids are broken down into glycerol and fatty acids (Figure 18.6).

In stage 2, the collection of product building blocks generated in stage 1 is further degraded to yield an even more limited set of simpler metabolic intermediates. The deamination of amino acids leaves -keto acid carbon skeletons. Several of these -keto acids are citric acid cycle intermediates and are fed directly into stage 3 catabolism via this cycle. Others are converted either to the three-carbon -keto acid pyruvate or to the acetyl groups of acetyl-coenzyme A (acetyl-CoA). Glucose and the glycerol from lipids also generate pyruvate, whereas the fatty-acids are broken into two-carbon units that appear as acetylCoA. Because pyruvate also gives rise to acetyl-CoA, we see that the degradation of macromolecular nutrients converges to a common end product, acetylCoA (Figure 18.6).

The combustion of the acetyl groups of acetyl-CoA by the citric acid cycle and oxidative phosphorylation to produce CO2 and H2O represents stage 3 of catabolism. The end products of the citric acid cycle, CO2 and H2O, are the ultimate waste products of aerobic catabolism. As we shall see in Chapter 20, the oxidation of acetyl-CoA during stage 3 metabolism generates most of the energy produced by the cell.

Anabolic Pathways Diverge, Synthesizing an Astounding Variety of Biomolecules from a Limited Set of Building Blocks

A rather limited collection of simple precursor molecules is sufficient to provide for the biosynthesis of virtually any cellular constituent, be it protein, nucleic acid, lipid, or polysaccharide. All of these substances are constructed from appropriate building blocks via the pathways of anabolism. In turn, the building blocks (amino acids, nucleotides, sugars, and fatty acids) can be generated from metabolites in the cell. For example, amino acids can be formed by amination of the corresponding -keto acid carbon skeletons, and pyruvate can be converted to hexoses for polysaccharide biosynthesis.

Amphibolic Intermediates

Certain of the central pathways of intermediary metabolism, such as the citric acid cycle, and many metabolites of other pathways have dual purposes—they serve in both catabolism and anabolism. This dual nature is reflected in the

amphi ● from the Greek for “on both sides” designation of such pathways as amphibolic rather than solely catabolic or anabolic. In any event, in contrast to catabolism—which converges to the common intermediate, acetyl-CoA—the pathways of anabolism diverge from a small group of simple metabolic intermediates to yield a spectacular variety of cellular constituents.

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Corresponding Pathways of Catabolism and

Anabolism Differ in Important Ways

The anabolic pathway for synthesis of a given end product usually does not precisely match the pathway used for catabolism of the same substance. Some of the intermediates may be common to steps in both pathways, but different enzymatic reactions and unique metabolites characterize other steps. A good example of these differences is found in a comparison of the catabolism of glucose to pyruvic acid by the pathway of glycolysis and the biosynthesis of glucose from pyruvate by the pathway called gluconeogenesis. The glycolytic pathway from glucose to pyruvate consists of 10 enzymes. Although it may seem efficient for glucose synthesis from pyruvate to proceed by a reversal of all 10 steps, gluconeogenesis uses only seven of the glycolytic enzymes in reverse, replacing the remaining three with four enzymes specific to glucose biosynthesis. In similar fashion, the pathway responsible for degrading proteins to amino acids differs from the protein synthesis system, and the oxidative degradation of fatty acids to two-carbon acetyl-CoA groups does not follow the same reaction path as the biosynthesis of fatty acids from acetyl-CoA.

Metabolic Regulation Favors Different Pathways for Oppositely Directed Metabolic Sequences

A second reason for different pathways serving in opposite metabolic directions is that such pathways must be independently regulated. If catabolism and anabolism passed along the same set of metabolic tracks, equilibrium considerations would dictate that slowing the traffic in one direction by inhibiting a particular enzymatic reaction would necessarily slow traffic in the opposite direction. Independent regulation of anabolism and catabolism can be accomplished only if these two contrasting processes move along different routes or, in the case of shared pathways, the rate-limiting steps serving as the points of regulation are catalyzed by enzymes that are unique to each opposing sequence (Figure 18.7).

Activation of one mode is accompanied by reciprocal inhibition of the other mode.

● Parallel pathways of catabolism and anabolism must differ in at least one metabolic step in order that they can be regulated independently. Shown here are two possible arrangements of opposing catabolic and anabolic sequences between A and P.

(a) The parallel sequences proceed via independent routes. (b) Only one reaction has two different enzymes, a catabolic one (E3) and its anabolic counterpart (E6). These provide sites for regulation.

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to NAD molecules, reducing them to NADH. A second proton accompanies these reactions, appearing in the overall equation as H (Figure 18.10). In turn, NADH is oxidized back to NAD when it transfers its reducing equivalents to electron acceptor systems that are part of the metabolic apparatus of the mitochondria. The ultimate oxidizing agent (e acceptor) is O2, becoming reduced to H2O.

Oxidation reactions are exergonic, and the energy released is coupled with the formation of ATP in a process called oxidative phosphorylation. The NAD –NADH system can be viewed as a shuttle that carries the electrons released from catabolic substrates to the mitochondria, where they are transferred to O2, the ultimate electron acceptor in catabolism. In the process, the free energy released is trapped in ATP. The NADH cycle is an important player in the transformation of the chemical energy of carbon compounds into the chemical energy of phosphoric anhydride bonds. Such transformations of energy from one form to another are referred to as energy transduction. Oxidative phosphorylation is one cellular mechanism for energy transduction. Chapter 21 is devoted to electron transport reactions and oxidative phosphorylation.

NADPH Provides the Reducing Power for Anabolic Processes

Whereas catabolism is fundamentally an oxidative process, anabolism is, by its contrasting nature, reductive. The biosynthesis of the complex constituents of the cell begins at the level of intermediates derived from the degradative pathways of catabolism; or, less commonly, biosynthesis begins with oxidized substances available in the inanimate environment, such as carbon dioxide. When the hydrocarbon chains of fatty acids are assembled from acetyl-CoA units, activated hydrogens are needed to reduce the carbonyl (CPO) carbon of acetylCoA into a OCH2O at every other position along the chain. When glucose is

● Hydrogen and electrons released in the course of oxidative catabolism are transferred as hydride ions to the pyridine nucleotide, NAD , to form NADH H in dehydrogenase reactions of the type

AH2 NAD 88n A NADH H

The reaction shown is catalyzed by alcohol dehydrogenase.

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Mutations Create Specific Metabolic Blocks

Genetics provides an approach to the identification of intermediate steps in metabolism that is somewhat analogous to inhibition. Mutation in a gene encoding an enzyme often results in an inability to synthesize the enzyme in an active form. Such a defect leads to a block in the metabolic pathway at the point where the enzyme acts, and the enzyme’s substrate accumulates. Such genetic disorders are lethal if the end product of the pathway is essential or if the accumulated intermediates have toxic effects. In microorganisms, however, it is often possible to manipulate the growth medium so that essential end products are provided. Then the biochemical consequences of the mutation can be investigated. Studies on mutations in genes of the filamentous fungus Neurospora crassa led G. W. Beadle and E. L. Tatum to hypothesize in 1941 that genes are units of heredity that encode enzymes (a principle referred to as the “one gene–one enzyme” hypothesis).

Isotopic Tracers as Metabolic Probes

Another widely used approach to the elucidation of metabolic sequences is to “feed” cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard: radioactive isotopes, such as 14C, and stable “heavy” isotopes, such as 18O or 15N (Table 18.3). Because the chemical behavior of isotopically labeled compounds is rarely distinguishable from that of their unlabeled counterparts, isotopes provide reliable “tags” for observing metabolic changes. The metabolic fate of a radioactively labeled substance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 18.13).

Table 18.3

Properties of Radioactive and Stable “Heavy” Isotopes Used

as Tracers in Metabolic Studies

*The relative natural abundance of a stable isotope is important because, in tracer studies, the amount of stable isotope is typically expressed in terms of atoms percent excess over the natural abundance of the isotope.