Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Stoichiometry and Energetics of Metabolic Transformations
Biosynthesis
Biosynthesis of Low-Molecular-Weight Compounds

In the introductory sections, we noted that most processes in the microbial world are characterized by 1) the utilization of substrates or nutrients, 2) Cell growth, and 3) the release of metabolic products into the environment. Biosynthesis is intimately connected with all three aspects of cellular activity. The demand for nutrients is determined by The Cell's requirement for precursor molecules, chemical energy reserves, and reducing equivalents. Some biosynthetic products are excreted by the cell into the surrounding medium. Finally, The rate of biosynthesis dictates the rate at which new cellular components are formed and, consequently, the overall rate of cell growth. Cellular growth rates vary over an exceptionally wide range; for instance, E. coli Bacteria can divide every 20 minutes, The life cycle of a rat Liver cell ranges from two to three months, and adult human Nerve Cells do not self-replicate at all. Even in the latter case, however, The Need for biosynthesis does not disappear; here it is essential for maintaining cellular viability and repairing damage.

For biosynthetic purposes, the cell utilizes chemical resources generated during catabolic processes. As a rule, synthetic reactions are thermodynamically unfavorable and proceed only when coupled with the Hydrolysis of ATP to ADP or AMP. The hydrolysis of the pyrophosphate (PP_i) formed in the latter case releases additional Free energy (∆G⁰' ≈ 7 kcal/mol), which is also harnessed to drive synthesis reactions. Because nutrients are generally more oxidized than the compounds required by the cell, biosynthesis of the latter also requires reducing equivalents.

Biologically important low-molecular-weight compounds are predominantly monomers utilized by the cell to construct Biopolymers. A total of roughly 70 different compounds are required for this purpose: 4 ribonucleotides, 4 deoxyribonucleotides, 20 Amino Acids, about 15 Monosaccharides, and about 20 Fatty acids and other lipid precursors. In addition, reactions of a second type must synthesize ATP, NAD, other carrier molecules, and Coenzymes. All these substances are referred to as central intermediary metabolites. In this section, we will examine some of the principal pathways of intermediary metabolite Biosynthesis and the Organization of these pathways.

Amino acids can be divided into four groups that differ in their chemical Structure and The Nature of their biosynthetic precursors. As we will see later in our Discussion of Metabolic Regulation, the synthesis of each amino acid group is carried out via a branched sequence of regulated reactions. The biosynthesis of all amino acids originates from intermediates of carbon METABOLISM (see the scheme in Fig. 5.11).

Living cells assimilate nitrogen by incorporating it into the amino acids glutamine and glutamic acid. Initially, the interaction of ammonia with α-ketoglutaric acid (an intermediate in The Tricarboxylic Acid Cycle) yields glutamic acid:

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Glutamic acid can bind another ammonium ion and be converted into glutamine:

FIG. 5.11. Amino acid families and their carbohydrate precursors. (Stent, G., & Calendar, R., Molecular Genetics. — Moscow: Mir, 1981.)

The second reaction, which proceeds with the consumption of metabolic energy, takes place under conditions of ammonia deficiency in the medium. In some bacteria, NADH-dependent direct amination of Pyruvate to Alanine occurs, while other bacteria are capable of converting fumarate into aspartate. A number of microorganisms assimilate nitrogen in the form of nitrate NO₃^- and free nitrogen N₂; however, it is now known that these nutrients are first transformed into ammonia, which is subsequently assimilated by cells via the pathways described above.

All Other Amino Acids are formed from glutamate, either using its carbon Skeleton or through The transfer of an amino group to other molecules. For example, glutamate is converted into Proline As a result of two successive enzyme-catalyzed reactions and one non-enzymatic hydrolytic reaction According to the overall equation:

C₅NH₈O₄- + ATP + 2(NADPH + H+) →

  →C₅NH₈O₂- + ADP + P_i + 2NADP+ + H₂O      (5.33)

In most organisms, alanine and aspartate are formed via glutamate Transamination:

Глутамат+оксалоацетат → а-кетоглутарат + аспартат

Глутамат+Пируват → а-кетоглутарат + Аланин

All transamination reactions occur exclusively in the presence of the coenzyme Pyridoxal phosphate, a derivative of vitamin B₆ (pyridoxal).

To conclude this Brief Overview of Amino acid biosynthesis, Fig. 5.12 illustrates several individual steps as well as the overall pathway for the biosynthesis of the aspartate family of amino acids. It is noteworthy here that the majority of these steps proceed with the involvement of ATP or NADPH. The dashed lines in this figure indicate regulatory mechanisms that will be discussed later.

A detailed examination of nucleotide biosynthetic pathways goes beyond The Scope of this book. The sources of precursors for these compounds are indicated in Fig. 5.13. Readers can find more detailed information in the literature.

Acetyl-CoA and glycerol serve as starting Materials in the Biosynthesis of Fatty acids and other lipid precursors. To further emphasize the involvement of ATP and NADPH in biosynthesis, we will examine in somewhat greater detail the biosynthetic pathway of palmitic acid, CH₃(CH₂)₁₄COOH, the most common of the fatty acids. The first step in this pathway is the carboxylation of acetyl-CoA to yield malonyl-CoA:

Fig. 5.12. Scheme of reactions wherein aspartate is converted into Lysine, homoserine, Methionine, Threonine, and isoleucine. (Reproduced with permission from: Wood W. B., Willson J. H., Benbow В. M., Hood L. E., Biochemistry, A Problems Approach, 2d ed p. 294, Benjamin/Cummings Publishing Co., Menlo Park, CA, 1981.)

Subsequently, palmitate is synthesized from seven molecules of malonyl-CoA and one molecule of acetyl-CoA. This process occurs in a stepwise manner, and its overall result can be expressed by the following equation:

Note that in this process, according to equation (5.34), one molecule of high-energy phosphate is consumed for every molecule of malonyl-CoA formed.

To conclude this brief overview of biosynthesis, let us examine the PATHWAYS OF GLUCOSE formation and related compounds. We already know that Glucose Catabolism products are crucial precursors in the BIOSYNTHESIS OF AMINO Acids, NUCLEOTIDES, and fatty acids. Consequently, organisms growing on other carbon sources, such as СО₂, must convert them into glucose or one of its immediate metabolic derivatives. Furthermore, when carbon compounds are in excess relative to other nutrients, many cells convert glucose into storage CARBOHYDRATES for subsequent use.

FIG. 5.13. Pathway for The conversion of precursor substances into nucleotides and deoxynucleotides. [Reproduced with permission from: Biochemistry of Bacterial Growth, Mandelstam J., McQuillan K. (eds.), 2d ed., p. 33, Blackwell Scientific Publications, Oxford, 1973.]

Gluconeogenesis refers to the Synthesis of glucose in chemotrophs. Interestingly, the synthesis of glucose from pyruvate involves all the intermediates typical of the EMP pathway. Even more importantly, all Enzymes catalyzing near-equilibrium reactions in the EMP pathway also catalyze reversible reactions in glucose biosynthesis. At the same time, the phosphorylation and dephosphorylation reactions in glucose biosynthesis differ somewhat from analogous reactions in Glycolysis and are catalyzed by different enzymes. These biosynthetic reactions, shown in Fig. 5.14 (from bottom to top), occur spontaneously and are accompanied by a decrease in free energy. The overall stoichiometry of gluconeogenesis indicates that the process as a whole is endergonic and is not simply the EMP pathway running in reverse (GTP — guanosine triphosphate):

2 Pyruvate + 4ATP + 2GTP + 2NADH + 2H⁺ + 4H₂О →

→ glucose + 2NAD⁺ + 2GDP + 4ADP + 6P_i      (5.36)

FIG. 5.14. Breakdown of glucose via The Glycolytic Pathway (top to bottom) and BIOSYNTHESIS OF GLUCOSE (bottom to top). These two metabolic pathways share a series of reactions that are particularly close to equilibrium. Meanwhile, non-equilibrium reactions are catalyzed by different enzymes (mostly allosteric) depending on their direction, allowing the Synthesis and Breakdown of glucose to be regulated independently. (Reproduced from Lehninger A., Bioenergetics, 2d ed., р. 129, W. A. Benjamin, Inc., Palo Alto, CA, 1974.)

The most important biosynthetic reaction is the synthesis of glucose from СО₂ in plants. Without this process, The Biosphere as we know it could not exist. Moreover, the scale and efficiency of these reactions determine the volume of renewable carbon-based resources that serve as fuels and feedstocks in biochemical and chemical processes.

The light reactions of Photosynthesis were described above: the absorption of light by chlorophyll and other pigments and the generation of an excited electron flow that produces ATP and NADPH. Next, we will examine the dark reactions of photosynthesis, so named because they proceed in the absence of light as long as the cell maintains a sufficient supply of ATP and NADPH.

The initial incorporation of СО₂ into the cycle occurs via a reaction with ribulose-1,5-diphosphate:

The glyceraldehyde-3-phosphate formed at this stage can subsequently undergo several transformations, notably entering a reaction sequence similar to gluconeogenesis to yield glucose. The Reactions of the Calvin cycle (Fig. 5.15) regenerate ribulose-1,5-diphosphate, which can then assimilate more СО₂ according to reaction (5.37). Unlike the TCA cycle, which essentially serves as a source of ATP and NADH, The Calvin Cycle consumes both ATP and NADPH. Since one molecule of СО₂ is utilized in a single turn of the Calvin cycle, the cycle must be repeated six times for the synthesis of one glucose molecule; the overall equation for glucose synthesis is then as follows:

6СО₂ + 12NADPH + 12Н⁺ + 18АТР + 12Н₂О →

→ С₆Н₁₂О₆ + 12NADP⁺ + 18ADP + 18Р_і      (5.38)