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

The Chemical Basis of Life
The Hierarchy of Cell Structure

In the previous sections, we examined the MAIN TYPES OF biologically important low-molecular-weight compounds and the Biopolymers constructed from them. Although we have repeatedly emphasized the relationship between the Chemical Structure of these substances and their Functions in The Cell, it is useful to discuss once again the dynamic nature and compartmentalization of these functions. As a living cell grows, all the biopolymers discussed in this chapter must be continuously synthesized. Typically, the cell's nutrient medium consists of sugars, carbon dioxide, Certain Amino Acids, Water, and A number of inorganic ions, while biopolymers and many of the monomers required for their construction are generally absent in any significant quantities. Thus, the cell must synthesize all other necessary amino acids, Nucleic Acids, Lipids, Proteins, and other substances from the simplest available precursors. The energy-consuming processes of precursor synthesis and biopolymer formation are discussed in Chapter 5.

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FIG. 2.27. Distribution of the biologically important substances described in this chapter in order of increasing structural complexity and level of Organization. (From: Lehninger A., Biochemistry. — Moscow: Mir, 1976, p. 22.)

A multitude of supramolecular structures exist within the cell. As we have already seen, The cell membrane, for example, is a complex combination of many types of molecules. Another example is Ribosomes, which are specific complexes of several different proteins and nucleic acids. In many cases, Enzymes that catalyze several consecutive Chemical Reactions are combined into a single multenzyme complex, which apparently ensures maximum efficiency in the utilization of intermediates. The final level of structural complexity, directly preceding the cell itself, is occupied by Organelles such as Mitochondria and METABOLISM/14.html">Chloroplasts. The various levels of complexity, from the simplest substances to the cell, are shown schematically in Fig. 2.27.

FIG. 2.28. Schematic diagram of the compartmentalization of the biologically important molecules described in this chapter within the E. coli cell.

A diagram of the localization of the various biologically important substances discussed in this chapter within a prokaryotic microorganism cell is shown in Fig. 2.28, and the overall Chemical composition of E. coli is summarized in Table 2.12. Small molecules such as Amino Acids and simple sugars, as well as some significantly larger molecules, such as certain enzymes and tRNAs, are uniformly distributed throughout the Cytoplasm. Other biopolymers are localized in specific regions within the cell or on its surface, such as the cell membrane.

Table 2.12. Composition of a rapidly growing E. coli cell^a

^a Watson J. D., Molecular Biology of the Gene. — Moscow: Mir, 1978, p. 70.

The connection between various ions, nutrients, intermediates, and other cellular components is maintained through an extensive network of chemical reactions (Chaps. 5 and 6). Individual reactions within this network are catalyzed by enzymes, which enable them to occur under mild conditions that, as shown above, favor the preservation of the Native State of proteins. A number of energy-consuming reactions (Chap. 5), along with the tendency of certain molecules to spontaneously self-assemble into localized structural elements within the cell, drive The formation of membranes, multenzyme complexes, and organelles.

Chapters 3 and 4 are devoted to the Kinetics of enzyme-catalyzed reactions and their Practical Applications. Furthermore, in Chapters 5–7, we will examine the energetics and stoichiometry of cellular processes, genetics, and the Regulation of cellular reaction networks, as well as mathematical expressions describing cell population growth kinetics. Following these introductory chapters, we will proceed to the main subject of the book: The Study of biochemical reactors.

Exercises

2.1. Chemical structure. a) Write the complete or generalized chemical formulas for the substances listed below. Explain the (possible) functional role of the various groups in these molecules.

1. Fat, phospholipid, micelle, vitamin A.

2. D(+)-Glucose, pyranose, lactose, starch, amylopectin, Cellulose.

3. Ribonucleotide, adenosine monophosphate (AMP), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD), deoxyribonucleic acid (DNA).

4. Amino acid, Lysine, tripeptide, protein.

5. Globular and Fibrous proteins.

б) Draw a diagram of the levels of Protein Structure, indicating the most important features of each level (from primary to quaternary structure).

2.2. Spectrophotometry. The intensity of Light absorption by a solution at specific wavelengths is measured using a spectrophotometer, an instrument of great importance in analytical biochemistry. Spectrophotometry is used to determine the concentrations of solutes, and they can be identified by The Nature of their absorption spectra. Spectrophotometric concentration determination is based on the Beer–Lambert law:

where A is the absorbance or optical density; I₀ is the intensity of the incident light; I is the intensity of the transmitted light; am is the molar extinction coefficient; b is the path length of the light beam; c is the molar concentration of the solute.

Typically, the wavelength of the incident light is chosen to be λ_max, at which the solute under study absorbs most strongly. Table 2Ex2.1 lists the λ_max values for a number of biologically important compounds and their corresponding molar extinction coefficients (at a light path length b = 1 cm and pH 7). Other necessary data can be found in reference books; note that λ_max values often lie in the ultraviolet region.

Table 2Ex2.1

a) An ATP solution in a 1 cm cuvette transmits 70% of the incident light at 259 nm. Calculate the concentration of ATP. What is the absorbance of a 5∙10^-5 M ATP solution?

b) Assume that compounds A and B have the same molar extinction coefficients given in Table 2У2.2. Calculate

Table 2У2.2

the concentrations of A and B in a solution containing both A and B and having an absorbance (in a 1 cm cuvette) of 0.35 at 340 nm and 0.220 at 410 nm.

2.3. Molecular masses. To estimate molecular mass by sedimentation and diffusion Methods, the Svedberg equation is frequently used:

where R is the gas constant; T is the absolute Temperature; s is the sedimentation coefficient, s; D is the diffusion coefficient, cm²/s; v is the partial specific volume of the substance, cm³/g; ρ is the density of the solution, g/cm³.

Verify the molecular masses of the substances listed in Table 2У3.1 (T = 20°C). Assume ρ = 1.0.

Table 2У3.1

2.4. Buffer capacity and pH of polymer solutions. a) Calculate the pH of 0.2 M solutions of Serine, serine hydrochloride, and serine potassium salt.

b) Draw The titration curve of a serine hydrochloride solution with a strong base (KOH); graduate the scales in pH values and the number of moles of KOH added per 1 mole of serine, respectively.

c) Calculate the pH of solutions of linear Polypeptides containing 5, 20, and 100 serine residues, their corresponding hydrochlorides, and potassium salts, at a weight percentage concentration equal to that of a 0.2 M serine hydrochloride solution. Assume that the dissociation constant is independent of the degree of polymerization.

d) Calculate the pH of a lysine monohydrochloride solution at a weight percentage concentration equal to that of a 0.2 M serine hydrochloride solution. Assume the equilibrium constants for the protonation of the α-amino group and the side-chain amino group are 8.91∙10^-6 and 2.95∙10^-11 L/mol, respectively.

e) Calculate the pH of 1% (by weight) solutions of cellulose and starch (assume that the molecular mass of starch is very large, >10⁶). In general, how do storage polymers affect cell pH?

2.5. Separation of amino acids. a) Evaluate whether the following statements are correct, and justify your Answers:

1. The pK of a simple amino acid is expressed by the formula

2. In the range between pK_COOH and pKNH₂, Amino acids are neutral.

b) Chromatographic Supports that are sulfonated polystyrene derivatives are successfully used in the chromatographic separation of amino acids. The basic groups of amino acids interact with negatively charged sulfonic acid groups, while the hydrophobic regions of amino acids interact with the aromatic moieties of the polymer. Estimate the approximate elution order of amino acids at pH 3, 7, and 10 from a Column containing:

1. Only sulfonic acid groups on an inert support.

2. Only polystyrene.

3. Sulfonated polystyrene with equally accessible sulfonic acid groups and phenyl rings.

c) Which interactions, hydrophilic or hydrophobic, will play a decisive role in the separation of a mixture of polypeptides and a mixture of proteins on the chromatographic supports listed in the previous exercise?

2.6. Physical chemistry of macromolecules. a) Using your knowledge, specialized chemistry literature, and other sources, describe the general chemical processes and/or physical conformational changes that accompany frying an egg, making dough, curdling milk, Setting jelly, blowing a soap bubble, protein crystallization, leather tanning, and whipping egg whites.

b) Important parameters of food substitutes from microbial and other sources, such as single-cell proteins and plant-based meat substitutes, are their degree of texturization and rheological properties (flow under applied force). Outline how you would organize a laboratory for testing synthetic food samples. (What training would you provide for the laboratory staff? What testing methods would you set up?)

2.7. Material balance equation; stoichiometry. Among the chemical elements, carbon, oxygen, hydrogen, and nitrogen are primarily required for cell growth. Assume that the sources of carbon and nitrogen are CH₂O and NH₄⁺, respectively:

a) Describe the constraints imposed on the coefficients a, β, etc., by the conservation of elemental composition. What additional information is required to determine all the coefficients for a given cell "composition"?

b) Complete the same task for a photosynthetic system:

c) Describe in detail a series of experiments by which you could determine the Elemental Composition of the cell (coefficients a, b, c, d).

2.8. Stoichiometry; aerobic and anaerobic microorganisms. To facilitate calculations of nutrient requirements, aeration rates, and Heat transfer (Chs. 5, 8), an apparent molecular formula can be assigned to the cell, and on this basis, a general stoichiometric equation for cell growth can be written.

a) Calculate the formula of a Yeast cell growing on a sugar-containing nutrient medium, if the following material balance has been experimentally determined for this process (assume that 1 "mole" of Cells is formed):

b) What formula should be assigned to the cell if 0.30017 "moles" of cells are formed in the above reaction?

c) During anaerobic Fermentation, a variety of partially oxidized compounds are typically formed along with the cell mass. Based on the "molecular formula" of the cell found in the previous exercise, calculate the unknown coefficients for the following typical reaction equations (here, the coefficients indicate the number of moles, not mass):

The rates of the processes described in Exercises 2.8a and 2.8c are easily controlled by varying the aeration rate, allowing yeast and ethanol to be produced in any intermediate ratio (the Vienna process) [Harrison J. B., Adv. Ind. Microbiol., 10, 129 (1971)].

2.9. Cellular and artificial lipid bilayer membranes. Table 2Ex9.1 compares the characteristics of natural and artificial bilayer lipid membranes.

Table 2Ex9.1^a

^a Tien H. Т., Bilayer Lipid Membranes: An Experimental Model for Introduction/36.html">Biological Membranes, in Chemistry of Biosurfaces, Hair M. L. (ed.), vol. 1, p. 239, Marcel Dekker, New York, 1971.

a) Why do the Two Types of membranes have approximately the same thickness? (Try to provide a detailed and well-reasoned answer.)

b) Which cellular functions are (likely) facilitated by the high resistance and high electrical capacitance of the membranes?

c) Compare the breakdown voltage of about 1 V/Å with The ratio of the ionization potential of any hydrocarbon containing 10 or more carbon atoms to its molecular length. Discuss the results.

2.10. Concerted transition model for oxygen binding by Hemoglobin. According to the concerted transition model, the inactive form of the protein T₀ is in equilibrium with the active form R₀:

In turn, the active form, which in the case of hemoglobin is composed of four subunits, can bind one molecule of substrate S with each subunit:

Let us denote the dissociation constant of the hemoglobin-substrate complex formed in each of the above stages by the symbol K_DS:

and calculate the ratios for various s/K_DS and L

Plot the dependence of y on s/K_Ds at L = 9000. Find a plot of The amount of O₂ bound by hemoglobin versus the partial pressure of O₂ in biochemical literature and compare it with your plot. In the literature found, read about other cooperative phenomena in biochemistry.

2.11. Multisubunit enzymes; error probability. At first glance, it might seem that enzymes composed of several subunits, such as allosteric enzymes, have a more complex structure than single-chain proteins. Nevertheless, the relative probability of error during The Biosynthesis of such proteins may turn out to be lower.

a) Consider the synthesis of two proteins, each composed of 850 amino acid residues. Let the first protein consist of a single polypeptide chain, and the second of three subunits with 200, 300, and 350 residues. Estimate the relative amount of the First and Second proteins with one or more errors, assuming that the error probability is the same for any residue and is 5∙10^-9 per residue, and that the presence of two errors in any chain prevents its further synthesis or incorporation into the active structure.

b) However convincing the calculations in Exercise 2.11a may be, it is known that in a number of enzymes, the Cleavage of a small part of the Amino Acid Sequence is not accompanied by a loss of activity, and that the same function can be performed by a fairly large number of proteins with different primary structures (see, for example, the various structures of Cytochromes c in the monograph: Lehninger A., Principles of Biochemistry, Vol. 1. — M.: Mir, 1985). What other functions might these seemingly nonessential Amino acid sequences and residues perform in the cell? (Recall the Internal Structure of the cell.)

2.12. Metabolism. To study the energy and mass balances of cellular processes, it is necessary to know the sequence in which chemical transformations occur and the cellular locations where each of them takes place. (We will study the Various metabolic pathways in more detail in subsequent chapters.) Based on the data presented in Figs. 2.27 and 2.28, draw a diagram of the formation of major biopolymers and higher Structural elements of the cell; use arrows to indicate The sequence of processes and the changes in their locations; assume that all structural elements of the cell are formed from simple precursors, such as O₂, H₂O, NH₄⁺, and glucose. Where necessary, indicate the presence (or absence) of spatial constraints for these metabolic intermediates.

2.13. Fitness of biologically important compounds. In this chapter, we have discussed monomeric compounds and biopolymers that play a crucial role in the life of microorganisms. Read book [12], which discusses, from the perspective of the planet's evolutionary history, why these particular compounds arose during evolution. List Blum's main arguments for the fitness of water, glucose, and ATP to perform their biological roles.

2.14. Gram stain. Of all the staining reactions in bacteriology, the Gram stain is the most frequently used. Copy the standard Gram Staining Procedure from any microbiology manual. Discuss the main steps of the procedure and The Mechanism of the Gram stain.

2.15. Intracellular concentrations. Calculate the molar concentrations of DNA, various RNAs, amino acids and their precursors, and lipids and their precursors in the E. coli cell. Determine the total molar concentration of substances in E. coli and discuss the applicability of thermodynamic laws for dilute (ideal) solutions to the E. coli cytoplasm.

2.16. Enzyme Structure. The Active Site of the enzyme carboxypeptidase, which contains 307 amino acid residues, includes Arginine (145), Tyrosine (248), and glutamic acid (270) (the numbers indicate the sequence position of The amino acid residue starting from the N-terminus of the chain). Assuming that the enzyme molecule has an alpha-helical structure, calculate the distances between these three residues. What structural changes necessary for the catalytic effect occur in the native carboxypeptidase molecule if they bring these three residues closer together to distances on the order of tenths of a nanometer?

2.17. Thermodynamics of protein polypeptide chain folding. Consider a hypothetical protein in an aqueous environment. Suppose that one part of this protein molecule has a defined Secondary structure, while another part contains Ser, Thr, Asn, and two nonpolar residues. Determine ∆G (kcal/mol of protein) and estimate whether the folding of the second part of the protein will occur under the following conditions:

a) All polar groups in the sequence form Hydrogen Bonds.

b) All polar groups, except for those in the side chains, form intramolecular hydrogen bonds.

c) None of the polar groups in the sequence participate in the formation of intramolecular hydrogen bonds.

Assume that The transfer of a hydrophobic residue from an aqueous medium at neutral pH to the nonpolar interior of the protein is accompanied by ∆G ≈ -4 kcal/mol (per residue) and that the formation of a Hydrogen bond between any two unbound polar groups has a ∆G ≈ -5 kcal/mol (per polar group). Note that residues not involved in intramolecular hydrogen bonds can form hydrogen bonds with water.

Table 2U18.1

2.18. Determination of protein amino acid sequence. Partial Hydrolysis of protein polypeptide chains yields fragments whose amino acid sequence can provide the necessary information about the Primary Structure of the entire protein. Table 2U18.1 lists a number of agents that specifically hydrolyze certain peptide bonds:

Another important tool for determining amino acid sequences is Sanger's reaction, in which 2,4-dinitrofluorobenzene reacts with the N-terminal amino acid to form the corresponding dinitrophenyl (DNP) derivative.

Table 2U18.2

Table 2U18.2 presents a set of experimental results obtained during the Study of the A chain of bovine Insulin. Determine its primary structure. Complete acid hydrolysis of the protein chain yields Gly, Ala, 2 Val, 2 Leu, Ile, 4 Cys, 2Asp, 4 Glu, 2 Ser, and 2 Tyr.

References

1. Stryer L., Biochemistry, vols. 1–3. — Moscow: Mir, 1984. An in-depth, highly readable textbook that covers fundamental biochemical concepts in the light of recent advances in molecular biology.

2. Yudkin M., Offord R., A Guidebook to Biochemistry, Cambridge University Press, London, 1971. A good introductory course that presents the basics of biochemistry with a minimum of detail.

3. Loewy A. G., Siekevitz P., Cell Structure and function. — Moscow: Mir, 1971. The third part of this book is an excellent short Introduction to Biochemistry; its particular strength is the significant emphasis placed on the experimental testing of biological theories and models.

4. Lehninger A. L., Biochemistry, 2nd ed., Worth Publishers, New York, 1975; Russian Translation of the first edition: Lehninger A., Biochemistry. — Moscow: Mir, 1976. An outstanding textbook for advanced study of biochemistry. Constant reference to Examples from cell and human physiology helps the reader better understand, memorize, and assimilate the material.

5. Lehninger A., Principles of Biochemistry, vols. 1–3. — Moscow: Mir, 1985. This relatively recent edition is intended for students; written in the same style as the previous book, it features a more detailed Discussion of current problems and future Prospects in molecular biology.

6. Mahler H. R., Cordes E. H., Biological Chemistry, Harper and Row, Publishers, Inc., New York, 1971. This book covers the chemical and mathematical aspects of biochemistry in greater detail than Lehninger's works; the sections on experimental methods in biochemistry and microbiology discuss modeling and transport phenomena more deeply and thoroughly than most other textbooks.

7. Watson J. D., Molecular Biology of the Gene. — Moscow: Mir, 1978. Although this well-written and now classic book focuses primarily on molecular biology, its introductory chapters briefly outline the fundamentals of biochemistry. It convincingly demonstrates The Importance of weak interactions and Molecular Dynamics to the structure and function of biochemically important compounds.

8. Wood W. B., Wilson J. H., Benbow R. M., Hood L. E., Biochemistry: A Problems Approach, 2nd ed., Benjamin/Cummings Publishing Co., Menlo Park, California, 1981. A concise Overview of the fundamentals of biochemistry, supplemented with exercises (and their answers).

9. Dowben R. M., General Physiology: A Molecular Approach, Harper and Row, Publishers, Inc., New York, 1969. This engagingly and fascinatingly written book focuses on cell physiology, viewed through the lens of physical chemistry principles.

10. Dickerson R. E., Geis I., The Structure and Action of Proteins, Harper and Row, Publishers, Inc., New York, 1969. Co-authored by a biologist and a science illustrator, this monograph offers a unique perspective on various Protein Functions, particularly the relationship between structure and function. It is a valuable companion to any of the books listed above.

11. Watson J. D., The Double Helix. — Moscow: Mir, 1969. The story of the Discovery of the DNA Structure, a turning point in The Development of biology. It also offers a candid look at how scientific discoveries are actually made in modern science.

12. Blum H. F., Time’s Arrow and Evolution, 3rd ed., Princeton University Press, Princeton, N.J., 1968. A study of the biological fitness of elements and compounds from an evolutionary perspective.

13. Fukui, Ishida, Microbial Production of Amino Acids, Kodansha Ltd., Tokyo, and John Wiley and Sons, Inc., New York, 1972. An interesting review of various methods for isolating metabolic intermediates, in this case, amino acids.