Chapter 6: Microbial Metabolism

Microbial growth requires the polymerization of biochemical building blocks into proteins, nucleic acids, polysaccharides, and lipids. The building blocks must come preformed in the growth medium or must be synthesized by the growing cells. Additional biosynthetic demands are placed by the requirement for coenzymes that participate in enzymatic catalysis. Biosynthetic polymerization reactions demand the transfer of anhydride bonds from adenosine triphosphate (ATP). Growth demands a source of metabolic energy for the synthesis of anhydride bonds and for the maintenance of transmembrane gradients of ions and metabolites.

Metabolism has two components, catabolism and anabolism (Figure 6-1). Catabolism encompasses processes that harvest energy released from the breakdown of compounds (eg, glucose), and using that energy to synthesize ATP. In contrast, anabolism, or biosynthesis, includes processes that utilize the energy stored in ATP to synthesize and assemble the subunits, or building blocks, of macromolecules that make up the cell. The sequence of building blocks within a macromolecule is determined in one of two ways. In nucleic acids and proteins, it is template-directed: DNA serves as the template for its own synthesis and for the synthesis of the various types of RNA; messenger RNA serves as the template for the synthesis of proteins. In carbohydrates and lipids, on the other hand, the arrangement of building blocks is determined entirely by enzyme specificities. Once the macromolecules have been synthesized, they self-assemble to form the supramolecular structures of the cell, eg, ribosomes, membranes, cell wall, flagella, and pili.

The rate of macromolecular synthesis and the activity of metabolic pathways must be regulated so that biosynthesis is balanced. All of the components required for macromolecular synthesis must be present for orderly growth, and control must be exerted so that the resources of the cell are not expended on products that do not contribute to growth or survival.

This chapter contains a review of microbial metabolism and its regulation. Microorganisms represent extremes of evolutionary divergence, and a vast array of metabolic pathways is found within the group. For example, any of more than half a dozen different metabolic pathways may be used for assimilation of a relatively simple compound, benzoate, and a single pathway for benzoate assimilation may be regulated by any of more than half a dozen control mechanisms. Our goal is to illustrate the principles that underlie metabolic pathways and their regulation. The primary principle that determines metabolic pathways is that they are achieved by organizing relatively few biochemical-type reactions in a specific order. Many biosynthetic pathways can be deduced by examining the chemical structures of the starting material, the end product, and perhaps one or two metabolic intermediates. The primary principle underlying metabolic regulation is that enzymes tend to be called into play only when their catalytic activity is required. The activity of an enzyme may be changed by varying either the amount of enzyme or the amount of substrate. In some cases, the activity of enzymes may be altered by the binding of specific effectors, metabolites that modulate enzyme activity.

Glucose 6-Phosphate and Carbohydrate Interconversions

The biosynthetic origins of building blocks and coenzymes can be traced to relatively few precursors, called focal metabolites. Figures 6-2, 6-3, 6-4, and 6-5 illustrate how the respective focal metabolites glucose 6-phosphate (G6PD), phosphoenolpyruvate, oxaloacetate, and α-ketoglutarate give rise to most biosynthetic end products.

Figure 6-2 illustrates how G6PD is converted to a range of biosynthetic end products via phosphate esters of carbohydrates with different chain lengths. Carbohydrates possess the empirical formula (CH₂O)_n, and the primary objective of carbohydrate metabolism is to change n, the length of the carbon chain. Mechanisms by which the chain lengths of carbohydrate phosphates are interconverted are summarized in Figure 6-6. In one case, oxidative reactions are used to remove a single carbon from G6PD, producing the pentose derivative ribulose 5-phosphate. Isomerase and epimerase reactions interconvert the most common biochemical forms of the pentoses: ribulose 5-phosphate, ribose 5-phosphate, and xylulose 5-phosphate. Transketolases transfer a two-carbon fragment from a donor to an acceptor molecule. These reactions allow pentoses to form or to be formed from carbohydrates of varying chain lengths. As shown in Figure 6-6, two pentose 5-phosphates (n = 5) are interconvertible with triose 3-phosphate (n = 3) and heptose 7-phosphate (n = 7); pentose 5-phosphate (n = 5) and tetrose 4-phosphate (n = 4) are interconvertible with triose 3-phosphate (n = 3) and hexose 6-phosphate (n = 6).

The six-carbon hexose chain of fructose 6-phosphate can be converted to two three-carbon triose derivatives by the consecutive action of a kinase and an aldolase on fructose 6-phosphate. Alternatively, aldolases, acting in conjunction with phosphatases, can be used to lengthen carbohydrate molecules: Triose phosphates give rise to fructose 6-phosphate; a triose phosphate and tetrose 4-phosphate form heptose 7-phosphate. The final form of carbohydrate chain length interconversion is the transaldolase reaction, which interconverts heptose 7-phosphate and triose 3-phosphate with tetrose 4-phosphate and hexose 6-phosphate.

The coordination of different carbohydrate rearrangement reactions to achieve an overall metabolic goal is illustrated by the hexose monophosphate shunt (Figure 6-7). This metabolic cycle is used by Cyanobacteria for the reduction of NAD⁺ (nicotinamide adenine dinucleotide) to NADH (reduced nicotinamide adenine dinucleotide), which serves as a reductant for respiration in the dark. Many organisms use the hexose monophosphate shunt to reduce NADP⁺ (nicotinamide adenine dinucleotide phosphate) to NADPH (reduced nicotinamide adenine dinucleotide phosphate), which is used for biosynthetic reduction reactions. The first steps in the hexose monophosphate shunt are the oxidative reactions that shorten six hexose 6-phosphates (abbreviated as six C₆ in Figure 6-7) to six pentose 5-phosphates (abbreviated six C₅). Carbohydrate rearrangement reactions convert the six C₅ molecules to five C₆ molecules so that the oxidative cycle may continue.

Clearly, all reactions for interconversion of carbohydrate chain lengths are not called into play at the same time. Selection of specific sets of enzymes, essentially the determination of the metabolic pathway taken, is dictated by the source of carbon and the biosynthetic demands of the cell. For example, a cell given triose phosphate as a source of carbohydrate will use the aldolase–phosphatase combination to form fructose 6-phosphate; the kinase that acts on fructose 6-phosphate in its conversion to triose phosphate would not be expected to be active under these circumstances. If demands for pentose 5-phosphate are high, as in the case of photosynthetic carbon dioxide assimilation, transketolases that can give rise to pentose 5-phosphates are very active.

In sum, G6PD can be regarded as a focal metabolite because it serves both as a direct precursor for metabolic building blocks and as a source of carbohydrates of varying length that are used for biosynthetic purposes. G6PD itself may be generated from other phosphorylated carbohydrates by selection of pathways from a set of reactions for chain length interconversion. The reactions chosen are determined by the genetic potential of the cell, the primary carbon source, and the biosynthetic demands of the organism. Metabolic regulation is required to ensure that reactions that meet the requirements of the organism are selected.

Formation and Utilization of Phosphoenolpyruvate

Triose phosphates, formed by the interconversion of carbohydrate phosphoesters, are converted to phosphoenolpyruvate by the series of reactions shown in Figure 6-8. Oxidation of glyceraldehyde 3-phosphate by NAD⁺ is accompanied by the formation of the acid anhydride bond on the one carbon of 1,3-diphosphoglycerate. This phosphate anhydride is transferred in a substrate phosphorylation to adenosine diphosphate (ADP), yielding an energy-rich bond in ATP. Another energy-rich phosphate bond is formed by dehydration of 2-phosphoglycerate to phosphoenolpyruvate; via another substrate phosphorylation, phosphoenolpyruvate can donate the energy-rich bond to ADP, yielding ATP and pyruvate. Thus, two energy-rich bonds in ATP can be obtained by the metabolic conversion of triose phosphate to pyruvate. This is an oxidative process, and in the absence of an exogenous electron acceptor, the NADH generated by oxidation of glyceraldehyde 3-phosphate must be oxidized to NAD⁺ by pyruvate or by metabolites derived from pyruvate. The products formed as a result of this process vary and, as described later in this chapter, can be used in the identification of clinically significant bacteria.

Formation of phosphoenolpyruvate from pyruvate requires a substantial amount of metabolic energy, and two anhydride ATP bonds invariably are invested in the process. Some organisms—Escherichia coli, for example—directly phosphorylate pyruvate with ATP, yielding adenosine monophosphate (AMP) and inorganic phosphate (P_i). Other organisms use two metabolic steps: One ATP pyrophosphate bond is invested in the carboxylation of pyruvate to oxaloacetate, and a second pyrophosphate bond (often carried by guanosine triphosphate [GTP] rather than ATP) is used to generate phosphoenolpyruvate from oxaloacetate.

Formation and Utilization of Oxaloacetate

As already described, many organisms form oxaloacetate by the ATP-dependent carboxylation of pyruvate. Other organisms, such as E coli, which form phosphoenolpyruvate directly from pyruvate, synthesize oxaloacetate by carboxylation of phosphoenolpyruvate.

Succinyl-CoA is a required biosynthetic precursor for the synthesis of porphyrins and other essential compounds. Some organisms form succinyl-CoA by reduction of oxaloacetate via malate and fumarate. These reactions represent a reversal of the metabolic flow observed in the conventional tricarboxylic acid cycle (see Figure 6-11).

Formation of α-Ketoglutarate From Pyruvate

Conversion of pyruvate to α-ketoglutarate requires a metabolic pathway that diverges and then converges (Figure 6-9). In one branch, oxaloacetate is formed by carboxylation of pyruvate or phosphoenolpyruvate. In the other branch, pyruvate is oxidized to acetyl-CoA. It is noteworthy that, regardless of the enzymatic mechanism used for the formation of oxaloacetate, acetyl-CoA is required as a positive metabolic effector for this process. Thus, the synthesis of oxaloacetate is balanced with the production of acetyl-CoA. Condensation of oxaloacetate with acetyl-CoA yields citrate. Isomerization of the citrate molecule produces isocitrate, which is oxidatively decarboxylated to α-ketoglutarate.

Growth With Acetate

Acetate is metabolized via acetyl-CoA, and many organisms possess the ability to form acetyl-CoA (Figure 6-10). Acetyl-CoA is used in the biosynthesis of α-ketoglutarate, and in most respiratory organisms, the acetyl fragment in acetyl-CoA is oxidized completely to carbon dioxide via the tricarboxylic acid cycle (Figure 6-11). The ability to use acetate as a net source of carbon, however, is limited to relatively few microorganisms and plants. Net synthesis of biosynthetic precursors from acetate is achieved by coupling reactions of the tricarboxylic acid cycle with two additional reactions catalyzed by isocitrate lyase and malate synthase. As shown in Figure 6-12, these reactions allow the net oxidative conversion of two acetyl moieties from acetyl-CoA to one molecule of succinate. Succinate may be used for biosynthetic purposes after its conversion to oxaloacetate, α-ketoglutarate, phosphoenolpyruvate, or G6PD.

Growth With Carbon Dioxide: The Calvin Cycle

Similar to plants and algae, a number of microbial species can use carbon dioxide as a sole source of carbon. In almost all of these organisms, the primary route of carbon assimilation is via the Calvin cycle, in which carbon dioxide and ribulose diphosphate combine to form two molecules of 3-phosphoglycerate (Figure 6-13A). 3-Phosphoglycerate is phosphorylated to 1,3-diphosphoglycerate, and this compound is reduced to the triose derivative, glyceraldehyde 3-phosphate. Carbohydrate rearrangement reactions (see Figure 6-6) allow triose phosphate to be converted to the pentose derivative ribulose 5-phosphate, which is phosphorylated to regenerate the acceptor molecule, ribulose 1,5-diphosphate (Figure 6-13B). Additional reduced carbon, formed by the reductive assimilation of carbon dioxide, is converted to focal metabolites for biosynthetic pathways.

Cells that can use carbon dioxide as a sole source of carbon are termed autotrophic, and the demands for this pattern of carbon assimilation can be summarized briefly as follows: In addition to the primary assimilatory reaction giving rise to 3-phosphoglycerate, there must be a mechanism for regenerating the acceptor molecule, ribulose 1,5-diphosphate. This process demands the energy-dependent reduction of 3-phosphoglycerate to the level of carbohydrate. Thus, autotrophy requires carbon dioxide, ATP, NADPH, and a specific set of enzymes.

Depolymerases

Many potential growth substrates occur as building blocks within the structure of biologic polymers. These large molecules are not readily transported across the cell membrane and often are affixed to even larger cellular structures. Many microorganisms elaborate extracellular depolymerases that hydrolyze proteins (ie, proteases), nucleic acids (ie, nucleases), polysaccharides (eg, amylase), and lipids (eg, lipases). The pattern of depolymerase activities can be useful in the identification of microorganisms.

Oxygenases

Many compounds in the environment are relatively resistant to enzymatic modification, and utilization of these compounds as growth substrates demands a special class of enzymes, oxygenases. These enzymes directly use the potent oxidant molecular oxygen as a substrate in reactions that convert a relatively intractable compound to a form in which it can be assimilated by thermodynamically favored reactions. The action of oxygenases is illustrated in Figure 6-14, which shows the role of two different oxygenases in the utilization of benzoate.

Reductive Pathways

Some microorganisms live in extremely reducing environments that favor chemical reactions that would not occur in organisms using oxygen as an electron acceptor. In these organisms, powerful reductants can be used to drive reactions that allow the assimilation of relatively intractable compounds. An example is the reductive assimilation of benzoate, a process in which the aromatic ring is reduced and opened to form the dicarboxylic acid pimelate. Further metabolic reactions convert pimelate to focal metabolites.

Nitrogen Assimilation

The reductive assimilation of molecular nitrogen, also referred to as nitrogen fixation, is required for continuation of life on our planet. Nitrogen fixation is accomplished by a variety of bacteria and Cyanobacteria using a multicomponent nitrogenase enzyme complex. Despite the variety of organisms capable of fixing nitrogen, the nitrogenase complex is similar in most of them (Figure 6-15). Nitrogenase is a complex of two enzymes—one enzyme (dinitrogenase reductase) contains iron and the other (dinitrogenase) contains iron and molybdenum. Together, these enzymes catalyze the following reaction:

Because of the high activation energy of breaking the very strong triple bond that joins two nitrogen atoms, this reductive assimilation of nitrogen demands a substantial amount of metabolic energy. Somewhere between 20 and 24 molecules of ATP are hydrolyzed as a single N₂ molecule is reduced to two molecules of NH₃.

Additional physiologic demands are placed by the fact that nitrogenase is readily inactivated by oxygen. Aerobic organisms that use nitrogenase have developed elaborate mechanisms to protect the enzyme against inactivation. Some form specialized cells in which nitrogen fixation takes place, and others have developed elaborate electron transport chains to protect nitrogenase against inactivation by oxygen. The most significant of these bacteria in agriculture are the Rhizobiaceae, organisms that fix nitrogen symbiotically in the root nodules of leguminous plants.

The capacity to use ammonia as a nitrogen source is widely distributed among organisms. The primary portal of entry of nitrogen into carbon metabolism is glutamate, which is formed by reductive amination of α-ketoglutarate. As shown in Figure 6-16, there are two biochemical mechanisms by which this can be achieved. One, the single-step reduction catalyzed by glutamate dehydrogenase (Figure 6-16A) is effective in environments in which there is an ample supply of ammonia. The other, a two-step process in which glutamine is an intermediate (Figure 6-16B), is used in environments in which ammonia is in short supply. The latter mechanism allows cells to invest the free energy formed by hydrolysis of a pyrophosphate bond in ATP into the assimilation of ammonia from the environment.

The amide nitrogen of glutamine, an intermediate in the two-step assimilation of ammonia into glutamate (see Figure 6-16B), is also transferred directly into organic nitrogen appearing in the structures of purines, pyrimidines, arginine, tryptophan, and glucosamine. The activity and synthesis of glutamine synthase are regulated by the ammonia supply and by the availability of metabolites containing nitrogen derived directly from the amide nitrogen of glutamine.

Most of the organic nitrogen in cells is derived from the α-amino group of glutamate, and the primary mechanism by which the nitrogen is transferred is transamination. The usual acceptor in these reactions is an α-keto acid, which is transformed to the corresponding α-amino acid. α-Ketoglutarate, the other product of the transamination reaction, may be converted to glutamate by reductive amination (see Figure 6-16).

Tracing the Structures of Biosynthetic Precursors: Glutamate and Aspartate

In many cases, the carbon skeleton of a metabolic end product may be traced to its biosynthetic origins. Glutamine, an obvious example, clearly is derived from glutamate (Figure 6-17). The glutamate skeleton in the structures of arginine and proline (see Figure 6-17) is less obvious but readily discernible. Similarly, the carbon skeleton of aspartate, directly derived from the focal metabolite oxaloacetate, is evident in the structures of asparagine, threonine, methionine, and pyrimidines (Figure 6-18). In some cases, different carbon skeletons combine in a biosynthetic pathway. For example, aspartate semialdehyde and pyruvate combine to form the metabolic precursors of lysine, diaminopimelic acid, and dipicolinic acid (Figure 6-19). The latter two compounds are found only in prokaryotes. Diaminopimelic acid is a component of peptidoglycan in the cell wall, and dipicolinic acid represents a major component of endospores.

Synthesis of Cell Wall Peptidoglycan

The structure of peptidoglycan is shown in Figure 2-15; the pathway by which it is synthesized is shown in simplified form in Figure 6-20A. The synthesis of peptidoglycan begins with the stepwise synthesis in the cytoplasm of UDP-N-acetylmuramic acid-pentapeptide. N-Acetylglucosamine is first attached to uridine diphosphate (UDP) and then converted to UDP-N-acetylmuramic acid by condensation with phosphoenolpyruvate and reduction. The amino acids of the pentapeptide are sequentially added, each addition catalyzed by a different enzyme and each involving the split of ATP to ADP + P_i.

The UDP-N-acetylmuramic acid-pentapeptide is attached to bactoprenol (a lipid of the cell membrane) and receives a molecule of N-acetylglucosamine from UDP. Some bacteria (eg, Staphylococcus aureus) form a pentaglycine derivative in a series of reactions using glycyl-tRNA as the donor; the completed disaccharide is polymerized to an oligomeric intermediate before being transferred to the growing end of a glycopeptide polymer in the cell wall.

Final cross-linking (Figure 6-20B) is accomplished by a transpeptidation reaction in which the free amino group of a pentaglycine residue displaces the terminal D-alanine residue of a neighboring pentapeptide. Transpeptidation is catalyzed by one of a set of enzymes called penicillin-binding proteins (PBPs). PBPs bind penicillin and other β-lactam antibiotics covalently, partly because of a structural similarity between these antibiotics and the pentapeptide precursor. Some PBPs have transpeptidase or carboxypeptidase activities, their relative rates perhaps controlling the degree of cross-linking in peptidoglycan (a factor important in cell septation).

The biosynthetic pathway is of particular importance in medicine because it provides a basis for the selective antibacterial action of several chemotherapeutic agents. Unlike their host cells, bacteria are not isotonic with the body fluids. Their contents are under high osmotic pressure, and their viability depends on the integrity of the peptidoglycan lattice in the cell wall being maintained throughout the growth cycle. Any compound that inhibits any step in the biosynthesis of peptidoglycan causes the wall of the growing bacterial cell to be weakened and the cell to lyse. The sites of action of several antibiotics are shown in Figure 6-20A and B.

Synthesis of Cell Envelope Lipopolysaccharide

The general structure of the antigenic lipopolysaccharide of gram-negative cell envelopes is shown in Figure 2-19. The biosynthesis of the repeating end-group, which gives the cell envelope its antigenic specificity, is shown in Figure 6-21. Note the resemblance to peptidoglycan synthesis: In both cases, a series of subunits is assembled on a lipid carrier in the membrane and then transferred to open ends of the growing polymer.

Synthesis of Extracellular Capsular Polymers

The capsular polymers, a few examples of which are listed in Table 2-2, are enzymatically synthesized from activated subunits. No membrane-bound lipid carriers have been implicated in this process. The presence of a capsule is often environmentally determined: Dextrans and levans, for example, can only be synthesized using the disaccharide sucrose (fructose–glucose) as the source of the appropriate subunit, and their synthesis thus depends on the presence of sucrose in the growth environment.

Synthesis of Reserve Food Granules

When nutrients are present in excess of the requirements for growth, bacteria convert certain of them to intracellular reserve food granules. The principal ones are starch, glycogen, poly-β-hydroxybutyrate, and volutin, which consists mainly of inorganic polyphosphate (see Chapter 2). The type of granule formed is species specific. The granules are degraded when exogenous nutrients are depleted.

As described in Chapter 5, there are two major metabolic mechanisms for generating the energy-rich acid pyrophosphate bonds in ATP: substrate phosphorylation (the direct transfer of a phosphate anhydride bond from an organic donor to ADP) and phosphorylation of ADP by inorganic phosphate. The latter reaction is energetically unfavorable and must be driven by a transmembrane electrochemical gradient, the proton motive force. In respiration, the electrochemical gradient is created from externally supplied reductant and oxidant. Energy released by transfer of electrons from the reductant to the oxidant through membrane-bound carriers is coupled to the formation of the transmembrane electrochemical gradient. In photosynthesis, light energy generates membrane-associated reductants and oxidants; the proton motive force is generated as these electron carriers return to the ground state. These processes are discussed below.

Pathways of Fermentation

A. Strategies for Substrate Phosphorylation

In the absence of respiration or photosynthesis, cells are entirely dependent on substrate phosphorylation for their energy: Generation of ATP must be coupled to chemical rearrangement of organic compounds. Many compounds can serve as fermentable growth substrates, and many pathways for their fermentation have evolved. These pathways have the following three general stages: (1) Conversion of the fermentable compound to the phosphate donor for substrate phosphorylation. This stage often contains metabolic reactions in which NAD⁺ is reduced to NADH. (2) Phospho-rylation of ADP by the energy-rich phosphate donor. (3) Metabolic steps that bring the products of the fermentation into chemical balance with the starting materials. The most frequent requirement in the last stage is a mechanism for oxidation of NADH, generated in the first stage of fermentation, to NAD⁺ so that the fermentation may proceed. In the following sections, examples of each of the three stages of fermentation are considered.

B. Fermentation of Glucose

The diversity of fermentative pathways is illustrated by consideration of some of the mechanisms used by microorganisms to achieve substrate phosphorylation at the expense of glucose. In principle, the phosphorylation of ADP to ATP can be coupled to either of two chemically balanced transformations:

The biochemical mechanisms by which these transformations are achieved vary considerably.

In general, the fermentation of glucose is initiated by its phosphorylation to G6PD. There are two mechanisms by which this can be achieved: (1) Extracellular glucose may be transported across the cytoplasmic membrane into the cell and then phosphorylated by ATP to yield G6PD and ADP. (2) In many microorganisms, extracellular glucose is phosphorylated as it is being transported across the cytoplasmic membrane by an enzyme system in the cytoplasmic membrane that phosphorylates extracellular glucose at the expense of phosphoenolpyruvate, producing intracellular G6PD and pyruvate. The latter process is an example of vectorial metabolism, a set of biochemical reactions in which both the structure and the location of a substrate are altered (see Chapter 2). It should be noted that the choice of ATP or phosphoenolpyruvate as a phosphorylating agent does not alter the ATP yield of fermentation because phosphoenolpyruvate is used as a source of ATP in the later stages of fermentation (see Figure 6-8).

C. The Embden-Meyerhof Pathway

This pathway (Figure 6-22), a commonly encountered mechanism for the fermentation of glucose, uses a kinase and an aldolase (see Figure 6-6) to transform the hexose (C₆) phosphate to two molecules of triose (C₃) phosphate. Four substrate phosphorylation reactions accompany the conversion of the triose phosphate to two molecules of pyruvate. Thus, taking into account the two ATP pyrophosphate bonds required to form triose phosphate from glucose, the Embden-Meyerhof pathway produces a net yield of two ATP pyrophosphate bonds. Formation of pyruvate from triose phosphate is an oxidative process, and the NADH formed in the first metabolic step (Figure 6-22) must be converted to NAD⁺ for the fermentation to proceed; two of the simpler mechanisms for achieving this goal are illustrated in Figure 6-23. Direct reduction of pyruvate by NADH produces lactate as the end product of fermentation and thus results in acidification of the medium. Alternatively, pyruvate may be decarboxylated to acetaldehyde, which is then used to oxidize NADH, resulting in production of the neutral product ethanol. The pathway taken is determined by the evolutionary history of the organism and, in some microorganisms, by the growth conditions.

D. The Entner-Doudoroff and Heterolactate Fermentations

Alternative pathways for glucose fermentation include some specialized enzyme reactions, and these are shown in Figure 6-24. The Entner-Doudoroff pathway diverges from other pathways of carbohydrate metabolism by a dehydration of 6-phosphogluconate followed by an aldolase reaction that produces pyruvate and triose phosphate (Figure 6-24A). The heterolactate fermentation and some other fermentative pathways depend upon a phosphoketolase reaction (Figure 6-24B) that phosphorolytically cleaves a ketosephosphate to produce acetyl phosphate and triose phosphate. The acid anhydride acetyl phosphate may be used to synthesize ATP or may allow the oxidation of two NADH molecules to NAD⁺ as it is reduced to ethanol.

The overall outlines of the respective Entner-Doudoroff and heterolactate pathways are shown in Figures 6-25 and 6-26. The pathways yield only a single molecule of triose phosphate from glucose, and the energy yield is correspondingly low: Unlike the Embden-Meyerhof pathway, the Entner-Doudoroff and heterolactate pathways yield only a single net substrate phosphorylation of ADP per molecule of glucose fermented. Why have the alternative pathways for glucose fermentation been selected in the natural environment? In answering this question, two facts should be kept in mind. First, in direct growth competition between two microbial species, the rate of substrate utilization can be more important than the amount of growth. Second, glucose is but one of many carbohydrates encountered by microorganisms in their natural environment. Pentoses, for example, can be fermented quite efficiently by the heterolactate pathway.

E. Additional Variations in Carbohydrate Fermentations

Pathways for carbohydrate fermentation can accommodate many more substrates than described here, and the end products may be far more diverse than suggested thus far. For example, there are numerous mechanisms for oxidation of NADH at the expense of pyruvate. One such pathway is the reductive formation of succinate. Many clinically significant bacteria form pyruvate from glucose via the Embden-Meyerhof pathway, and they may be distinguished on the basis of reduction products formed from pyruvate, reflecting the enzymatic constitution of different species. The major products of fermentation, listed in Table 6-1, form the basis for many diagnostic tests used in the clinical laboratory.

F. Fermentation of Other Substrates

Carbohydrates are by no means the only fermentable substrates. Metabolism of amino acids, purines, and pyrimidines may allow substrate phosphorylations to occur. For example, arginine may serve as an energy source by giving rise to carbamoyl phosphate, which can be used to phosphorylate ADP to ATP. Some organisms ferment pairs of amino acids, using one as an electron donor and the other as an electron acceptor.

Patterns of Respiration

Respiration requires a closed membrane. In bacteria, the membrane is the cell membrane. Electrons are passed from a chemical reductant to a chemical oxidant through a specific set of electron carriers within the membrane, and as a result, the proton motive force is established (Figure 6-27); return of protons across the membrane is coupled to the synthesis of ATP. As suggested in Figure 6-27, the biologic reductant for respiration frequently is NADH, and the oxidant often is oxygen.

Tremendous microbial diversity is exhibited in the sources of reductant used to generate NADH, and many microorganisms can use electron acceptors other than oxygen. Organic growth substrates are converted to focal metabolites that may reduce NAD⁺ to NADH either by the hexose monophosphate shunt (see Figure 6-7) or by the tricarboxylic acid cycle (see Figure 6-11). Additional reductant may be generated during the breakdown of some growth substrates, such as fatty acids (see Figure 6-10).

Some bacteria, called chemolithotrophs, are able to use inorganic reductants for respiration. These energy sources include hydrogen, ferrous iron, and several reduced forms of sulfur and nitrogen. ATP derived from respiration and NADPH generated from the reductants can be used to drive the Calvin cycle (see Figure 6-13).

Compounds and ions other than O₂ may be used as terminal oxidants in respiration. This ability, ie, anaerobic respiration, is a widespread microbial trait. Suitable electron acceptors include nitrate, sulfate, and carbon dioxide. Respiratory metabolism dependent on carbon dioxide as an electron acceptor is a property found among representatives of a large microbial group, the archaebacteria. Representatives of this group possess, for example, the ability to reduce carbon dioxide to acetate as a mechanism for generating metabolic energy.

Bacterial Photosynthesis

Photosynthetic organisms use light energy to separate electronic charge to create membrane-associated reductants and oxidants as a result of a photochemical event. Transfer of electrons from the reductant to the oxidant creates a proton motive force. Many bacteria carry out a photosynthetic metabolism that is entirely independent of oxygen. Light is used as a source of metabolic energy, and carbon for growth is derived either from organic compounds (photoheterotroph) or from a combination of an inorganic reductant (eg, thiosulfate) and carbon dioxide (photolithotroph). These bacteria possess a single photosystem that, although sufficient to provide energy for the synthesis of ATP and for the generation of essential transmembrane ionic gradients, does not allow the highly exergonic reduction of NADP⁺ at the expense of water. This process, essential for oxygen-evolving photosynthesis, rests upon additive energy derived from the coupling of two different photochemical events driven by two independent photochemical systems. Among prokaryotes, this trait is found solely in the Cyanobacteria (blue-green bacteria). Among eukaryotic organisms, the trait is shared by algae and plants in which the essential energy-providing organelle is the chloroplast.

In their normal environment, microbial cells generally regulate their metabolic pathways so that no intermediate is made in excess. Each metabolic reaction is regulated not only with respect to all others in the cell but also with respect to the concentrations of nutrients in the environment. Thus, when a sporadically available carbon source suddenly becomes abundant, the enzymes required for its catabolism increase in both amount and activity; conversely, when a building block (eg, an amino acid) suddenly becomes abundant, the enzymes required for its biosynthesis decrease in both amount and activity.

The regulation of enzyme activity as well as enzyme synthesis provides both fine control and coarse control of metabolic pathways. For example, the inhibition of enzyme activity by the end product of a pathway constitutes a mechanism of fine control because the flow of carbon through that pathway is instantly and precisely regulated. The inhibition of enzyme synthesis by the same end product, on the other hand, constitutes a mechanism of coarse control. The preexisting enzyme molecules continue to function until they are diluted out by further cell growth, although unnecessary protein synthesis ceases immediately.

The mechanisms by which the cell regulates enzyme activity are discussed in the following section. The regulation of enzyme synthesis is discussed in Chapter 7.

The Regulation of Enzyme Activity

A. Enzymes as Allosteric Proteins

In many cases, the activity of an enzyme catalyzing an early step in a metabolic pathway is inhibited by the end product of that pathway. Such inhibition cannot depend on competition for the enzyme’s substrate binding site, however, because the structures of the end product and the early intermediate (substrate) are usually quite different. Instead, inhibition depends on the fact that regulated enzymes are allosteric: Each enzyme possesses not only a catalytic site, which binds substrate, but also one or more other sites that bind small regulatory molecules, or effectors. The binding of an effector to its site causes a conformational change in the enzyme such that the affinity of the catalytic site for the substrate is reduced (allosteric inhibition) or increased (allosteric activation).

Allosteric proteins are usually oligomeric. In some cases, the subunits are identical, each subunit possessing both a catalytic site and an effector site; in other cases, the subunits are different, one type possessing only a catalytic site and the other only an effector site.

B. Feedback Inhibition

The general mechanism that has evolved in microorganisms for regulating the flow of carbon through biosynthetic pathways is the most efficient that one can imagine. The end product in each case allosterically inhibits the activity of the first—and only the first—enzyme in the pathway. For example, the first step in the biosynthesis of isoleucine not involving any other pathway is the conversion of l-threonine to α-ketobutyric acid, catalyzed by threonine deaminase. Threonine deaminase is allosterically and specifically inhibited by l-isoleucine and by no other compound (Figure 6-28); the other four enzymes of the pathway are not affected (although their synthesis is repressed).

C. Allosteric Activation

In some cases, it is advantageous to the cell for an end product or an intermediate to activate rather than inhibit a particular enzyme. In the breakdown of glucose by E coli, for example, overproduction of the intermediates G6PD and phosphoenolpyruvate signals the diversion of some glucose to the pathway of glycogen synthesis; this is accomplished by the allosteric activation of the enzyme converting glucose 1-phosphate to ADP-glucose (Figure 6-29).

D. Cooperativity

Many oligomeric enzymes, possessing more than one substrate binding site, show cooperative interactions of substrate molecules. The binding of substrate by one catalytic site increases the affinity of the other sites for additional substrate molecules. The net effect of this interaction is to produce an exponential increase in catalytic activity in response to an arithmetic increase in substrate concentration.

E. Covalent Modification of Enzymes

The regulatory properties of some enzymes are altered by covalent modification of the protein. For example, the response of glutamine synthetase to metabolic effectors is altered by adenylylation, the covalent attachment of ADP to a specific tyrosyl side chain within each enzyme subunit. The enzymes controlling adenylylation also are controlled by covalent modification. The activity of other enzymes is altered by their phosphorylation.

F. Enzyme Inactivation

The activity of some enzymes is removed by their hydrolysis. This process can be regulated and sometimes is signaled by covalent modification of the enzyme targeted for removal.

• Metabolism consists of two components, catabolism and anabolism. Catabolism consists of processes that harvest energy from the breakdown of compounds and using that energy to synthesize ATP. Anabolism (or biosynthesis) consists of processes that use the energy stored in ATP to synthesize the subunits (or building blocks) of macromolecules that make up the cell.

• The biosynthetic origins of the building blocks can be traced to relatively few precursors, called focal metabolites.

• Peptidoglycan biosynthesis is unique to bacteria. Some antibiotics kill bacteria by selectively inhibiting steps in peptidoglycan biosynthesis.

• The Embden-Meyerhof, Entner-Doudoroff, and heterolactate pathways are three pathways used for glucose catabolism in bacteria. The pattern of end products is a characteristic used in the identification of bacterial species.

• In the absence of respiration or photosynthesis, bacteria are entirely dependent on substrate phosphorylation for their energy.

• Reductive assimilation of molecular nitrogen (or nitrogen fixation) is required for continuation of life on our planet. It is an energy-intensive process accomplished by a variety of bacteria and Cyanobacteria using a multicomponent nitrogenase enzyme complex.

• The regulation of enzyme activity provides both fine control and coarse control of metabolic pathways so that no intermediate is made in excess.