.
Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are prominent carriers of high-energy electrons. In NAD+, R = H; in NADP+, R = PO32-.
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At first glance, metabolism appears intimidating because of the sheer number of reactants and reactions. Nevertheless, there are unifying themes that make the comprehension of this complexity more manageable. These unifying themes include common metabolites, reactions, and regulatory schemes that stem from a common evolutionary heritage.
We have seen that phosphoryl transfer can be used to drive otherwise endergonic reactions, alter the energy of conformation of a protein, or serve as a signal to alter the activity of a protein. The phosphoryl-group donor in all of these reactions is ATP. In other words, ATP is an activated carrier of phosphoryl groups because phosphoryl transfer from ATP is an exergonic process. The use of activated carriers is a recurring motif in biochemistry, and we will consider several such carriers here.
Activated carriers of electrons for fuel oxidation. In aerobic organisms, the ultimate electron acceptor in the oxidation of fuel molecules is O2. However, electrons are not transferred directly to O2. Instead, fuel molecules transfer electrons to special carriers, which are either pyridine nucleotides or flavins. The reduced forms of these carriers then transfer their highpotential electrons to O2.
Nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) are prominent carriers of high-energy electrons. In NAD+, R = H; in NADP+, R = PO32-.
). The reactive part of NAD+ is its nicotinamide ring, a pyridine derivative synthesized from the vitamin niacin. In the oxidation of a substrate, the nicotinamide ring of NAD+ accepts a hydrogen ion and two electrons, which are equivalent to a hydride ion. The reduced form of this carrier is called NADH. In the oxidized form, the nitrogen atom carries a positive charge, as indicated by NAD+. NAD+ is the electron acceptor in many reactions of the type
In this dehydrogenation, one hydrogen atom of the substrate is directly transferred to NAD+, whereas the other appears in the solvent as a proton. Both electrons lost by the substrate are transferred to the nicotinamide ring.
This electron carrier consists of a flavin mononucleotide (FMN) unit (shown in blue) and an AMP unit (shown in black).
). The abbreviations for the oxidized and reduced forms of this carrier are FAD and FADH2, respectively. FAD is the electron acceptor in reactions of the type
The electrons and protons are carried by the isoalloxazine ring component of FAD and FADH2.
). FAD, like NAD+, can accept two electrons. In doing so, FAD, unlike NAD+, takes up two protons. These carriers of high-potential electrons as well as flavin mononucleotide (FMN), an electron carrier related to FAD, will be considered further in Chapter 18.An activated carrier of electrons for reductive biosynthesis. High-potential electrons are required in most biosyntheses because the precursors are more oxidized than the products. Hence, reducing power is needed in addition to ATP. For example, in the biosynthesis of fatty acids, the keto group of an added two-carbon unit is reduced to a methylene group in several steps. This sequence of reactions requires an input of four electrons.
). NADPH differs from NADH in that the 2′-hydroxyl group of its adenosine moiety is esterified with phosphate. NADPH carries electrons in the same way as NADH. However, NADPH is used almost exclusively for reductive biosyntheses, whereas NADH is used primarily for the generation of ATP. The extra phosphoryl group on NADPH is a tag that enables enzymes to distinguish between high-potential electrons to be used in anabolism and those to be used in catabolism.An activated carrier of two-carbon fragments. Coenzyme A, another central molecule in metabolism, is a carrier of acyl groups (Figure 14.16
). Acyl groups are important constituents both in catabolism, as in the oxidation of fatty acids, and in anabolism, as in the synthesis of membrane lipids. The terminal sulfhydryl group in CoA is the reactive site. Acyl groups are linked to CoA by thioester bonds. The resulting derivative is called an acyl CoA. An acyl group often linked to CoA is the acetyl unit; this derivative is called acetyl CoA. The ΔG°′ for the hydrolysis of acetyl CoA has a large negative value:
The hydrolysis of a thioester is thermodynamically more favorable than that of an oxygen ester because the electrons of the C=O bond cannot form resonance structures with the C—S bond that are as stable as those that they can form with the C—O bond. Consequently, acetyl CoA has a high acetyl potential (acetyl group-transfer potential) because transfer of the acetyl group is exergonic. Acetyl CoA carries an activated acetyl group, just as ATP carries an activated phosphoryl group.
The use of activated carriers illustrates two key aspects of metabolism. First, NADH, NADPH, and FADH2 react slowly with O2 in the absence of a catalyst. Likewise, ATP and acetyl CoA are hydrolyzed slowly (in times of many hours or even days) in the absence of a catalyst. These molecules are kinetically quite stable in the face of a large thermodynamic driving force for reaction with O2 (in regard to the electron carriers) and H2O (in regard to ATP and acetyl CoA). The kinetic stability of these molecules in the absence of specific catalysts is essential for their biological function because it enables enzymes to control the flow of free energy and reducing power.
| Carrier molecule in activated form | Group carried | Vitamin precursor |
|---|---|---|
| ATP | Phosphoryl | |
| NADH and NADPH | Electrons | Nicotinate (niacin) |
| FADH2 | Electrons | Riboflavin (vitamin B2) |
| FMNH2 | Electrons | Riboflavin (vitamin B2) |
| Coenzyme A | Acyl | Pantothenate |
| Lipoamide | Acyl | |
| Thiamine pyrophosphate | Aldehyde | Thiamine (vitamin B1) |
| Biotin | CO2 | Biotin |
| Tetrahydrofolate | One-carbon units | Folate |
| S-Adenosylmethionine | Methyl | |
| Uridine diphosphate glucose | Glucose | |
| Cytidine diphosphate diacylglycerol | Phosphatidate | |
| Nucleoside triphosphates | Nucleotides |
Note: Many of the activated carriers are coenzymes that are derived from water-soluble vitamins (Section 8.6.1).
| Type of reaction | Description |
|---|---|
| Oxidation-reduction | Electron transfer |
| Ligation requiring ATP cleavage | Formation of covalent bonds (i.e., carbon-carbon bonds) |
| Isomerization | Rearrangement of atoms to form isomers |
| Group transfer | Transfer of a functional group from one molecule to another |
| Hydrolytic | Cleavage of bonds by the addition of water |
| Addition or removal of functional groups | Addition of functional groups to double bonds or their removal to form double bonds |
Oxidation-reduction reactions are essential components of many pathways. Useful energy is often derived from the oxidation of carbon compounds. Consider the following two reactions:
These two oxidation-reduction reactions are components of the citric acid cycle (Chapter 17), which completely oxidizes the activated two-carbon fragment of acetyl CoA to two molecules of CO2. In reaction 1, FADH2 carries the electrons, whereas, in reaction 2, electrons are carried by NADH. In biosynthetic oxidation-reduction reactions, NADPH is the reductant.
Ligation reactions form bonds by using free energy from ATP cleavage. Reaction 3 illustrates the ATP-dependent formation of a carbon-carbon bond, necessary to combine smaller molecules to form larger ones. Oxaloacetate is formed from pyruvate and CO2.
The oxaloacetate can be used in the citric acid cycle or converted into amino acids such as aspartic acid.
Isomerization reactions rearrange particular atoms within the molecule. Their role is often to prepare a molecule for subsequent reactions such as the oxidation-reduction reactions described in point 1.
Reaction 4 is, again, a component of the citric acid cycle. This isomerization prepares the molecule for subsequent oxidation and decarboxylation by moving the hydroxyl group of citrate from a tertiary to a secondary position.
Group-transfer reactions play a variety of roles. Reaction 5 is representative of such a reaction. A phosphoryl group is transferred from the activated phosphoryl-group carrier, ATP, to glucose. This reaction traps glucose in the cell so that further catabolism can take place.
We saw earlier that group-transfer reactions are used to synthesize ATP (14.2.1). We will also see examples of their use in signaling pathways (Chapter 15).
Hydrolytic reactions cleave bonds by the addition of water. Hydrolysis is a common means employed to break down large molecules, either to facilitate further metabolism or to reutilize some of the components for biosynthetic purposes. Proteins are digested by hydrolytic cleavage (Chapters 9 and 10). Reaction 6 illustrates the hydrolysis of a peptide to yield two smaller peptides.
The addition of functional groups to double bonds or the removal of groups to form double bonds constitutes the final class of reactions. The enzymes that catalyze these types of reaction are classified as lyases (Section 8.1.3). An important example, illustrated in reaction 7, is the conversion of the six-carbon molecule fructose 1,6-bisphosphate (F-1, 6-BP) into 2 three-carbon fragments: dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.
This reaction is a key step in glycolysis, a key pathway for extracting energy from glucose (Section 16.1.3). Dehydrations to form double bonds, such as the formation of phosphoenolpyruvate (Table 14.1) from 2-phosphoglycerate (reaction 8), are important reactions of this type.
The dehydration sets up the next step in the pathway, a group-transfer reaction that uses the that uses the high phosphoryl transfer potential of the product PEP to form ATP from ADP.
Some metabolic pathways have similar sequences of reactions in common—in this case, an oxidation, the addition of a functional group (from a water molecule) to a double bond, and another oxidation. ACP designates acyl carrier protein.
. The same sequence of reactions is employed in the citric acid cycle, fatty acid degradation, the degradation of the amino acid lysine, and (in reverse) the biosynthesis of fatty acids. An effective way to learn is to look for commonalties in the diverse metabolic pathways that we will be studying. There is a chemical logic that, when exposed, renders the complexity of the chemistry of living systems more manageable and reveals its elegance.
It is evident that the complex network of reactions constituting intermediary metabolism must be rigorously regulated. At the same time, metabolic control must be flexible, because the external environments of cells are not constant. Metabolism is regulated by controlling (1) the amounts of enzymes, (2) their catalytic activities, and (3) the accessibility of substrates. The amount of a particular enzyme depends on both its rate of synthesis and its rate of degradation. The level of most enzymes is adjusted primarily by changing the rate of transcription of the genes encoding them. In E. coli, the presence of lactose, for example, induces within minutes a more than 50-fold increase in the rate of synthesis of β-galactosidase, an enzyme required for the breakdown of this disaccharide.
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. The inhibition of aspartate transcarbamoylase by cytidine triphosphate (Section 10.1) is a well-understood example of feedback inhibition. This type of control can be almost instantaneous. Another recurring mechanism is reversible covalent modification. For example, glycogen phosphorylase, the enzyme catalyzing the breakdown of glycogen, a storage form of sugar, is activated by phosphorylation of a particular serine residue when glucose is scarce (Section 21.2.1).
Hormones coordinate metabolic relations between different tissues, often by regulating the reversible modification of key enzymes. Hormones such as epinephrine trigger signal transduction cascades that lead to highly amplified changes in metabolic patterns in target tissues such as the muscle (Section 15.0.1). The hormone insulin promotes the entry of glucose into many kinds of cells. As will be discussed again in Chapter 15, many hormones act through intracellular messengers, such as cyclic AMP and calcium ion, that coordinate the activities of many target proteins.
Controlling the flux of substrates also regulates metabolism. The transfer of substrates from one compartment of a cell to another (e.g., from the cytosol to mitochondria) can serve as a control point.
An important general principle of metabolism is that biosynthetic and degradative pathways are almost always distinct. This separation is necessary for energetic reasons, as will be evident in subsequent chapters. It also facilitates the control of metabolism. In eukaryotes, metabolic regulation and flexibility also are enhanced by compartmentalization. For example, fatty acid oxidation takes place in mitochondria, whereas fatty acid synthesis takes place in the cytosol. Compartmentalization segregates opposed reactions.
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 proportional to the mole fraction of ATP plus half the mole fraction of ADP, given that ATP contains two anhydride bonds, whereas ADP contains one. Hence, the energy charge is defined as
High concentrations of ATP inhibit the relative rates of a typical ATP-generating (catabolic) pathway and stimulate the typical ATP-utilizing (anabolic) pathway.
). It is evident that the control of these pathways has evolved to maintain the energy charge within rather narrow limits. In other words, the energy charge, like the pH of a cell, is buffered. The energy charge of most cells ranges from 0.80 to 0.95. An alternative index of the energy status is the phosphorylation potential, which is defined as
The phosphorylation potential, in contrast with the energy charge, depends on the concentration of Pi and is directly related to the free energy-storage available from ATP.
How did the complex pathways that constitute metabolism evolve? This question, a difficult one to address, was approached in Chapter 2. The current thinking is that RNA was an early biomolecule and that, in an early RNA world, RNA served as catalysts and information-storage molecules (Section 2.2.2).
This fundamental building block is present in key molecules such as ATP, NADH, FAD, and coenzyme A. The adenine unit is shown in blue, the ribose unit in red, and the diphosphate unit in yellow.
)? A possible explanation is that these molecules evolved from the early RNA catalysts. Non-RNA units such as the isoalloxazine ring may have been recruited to serve as efficient carriers of activated electrons and chemical units, a function not readily performed by RNA itself. We can picture the adenine ring of FADH2 binding to a uracil unit in a niche of an RNA enzyme (ribozyme) by base-pairing, whereas the isoalloxazine ring protrudes and functions as an electron carrier. When the more versatile proteins replaced RNA as the major catalysts, the ribonucleotide coenzymes stayed essentially unchanged because they were already well suited to their metabolic roles. The nicotin amide unit of NADH, for example, can readily transfer electrons irrespective of whether the adenine unit interacts with a base in an RNA enzyme or with amino acid residues in a protein enzyme. With the advent of protein enzymes, these important cofactors evolved as free molecules without losing the adenosine diphosphate vestige of their RNA-world ancestry. That molecules and motifs of metabolism are common to all forms of life testifies to their common origin and to the retention of functioning modules through billions of years of evolution. Our understanding of metabolism, like that of other biological processes, is enriched by inquiry into how these beautifully integrated patterns of reactions came into being.
