Biochemical Processes Molecular Biology

Chapter 37

Biochemical Processes Molecular Biology

Biochemistry, molecular biology, and organic chemistry

• In the past four chapters, we have learned something about fats, carbohydrates, and proteins: their structures and how these are determined, and the kind of reactions they undergo in the test tube. These, we said, are biomolecules: they are participants in the chemical process we call life. But just what do they do! What reactions do they undergo, not in the test tube, but in a living organism?

• Even a vastly simplified answer to that question would fill and does a book as big as this one. Having come this far, though, we cannot help being curious. And so, in this chapter, we shall take a brief glance at the answer or, rather, at the kind of thing the answer entails.

• We shall look at just a few examples of biochemical processes: how one enzyme of the thousands in our bodies may work; what happens in one of the dozens of reactions by which carbohydrates are oxidized to furnish energy; how one kind of chemical compound fatty acids is synthesized. Finally, we shall learn a little about another class of biomolecules, the nucleic acids, and how they are involved in the most fascinating biochemical process of all heredity.

• The study of nucleic acids has become known as "molecular biology." Actually, of course, all of these processes are a part of molecular biology biology on the molecular level, and they are, in the final analysis, organic chemistry. And it is as organic chemistry that we shall treat them. We shall see how all these vital processes even the mysterious powers of enzymes come down to a matter of molecular structure as we know it: to molecular size and shape; to intermolecular and intramolecular forces; to the chemistry of functional groups; to acidity and basicity, oxidation and reduction; to energy changes and rate of reaction.

Mechanism of enzyme action. Chymotrypsin

• Enzymes, we have said, are proteins that act as enormously effective catalysts for biological reactions. To get some idea of how they work, let us examine the action of just one: chymotrypsin, a digestive enzyme whose job is to promote hydrolysis of certain peptide links in proteins. The sequence of the 245 amino acid residues in chymotrypsin has been determined and, through x-ray analysis, the conformation of the molecule is known (Fig. 37.1). It is, like all enzymes, a soluble globular protein coiled in the way that turns its hydrophobic parts inward, away from water, and that permits maximum intramolecular hydrogen bonding.

• The action of chymotrypsin has been more widely explored than that of any other enzyme. In crystalline form, it is available for studies in the test tube under a variety of conditions. It catalyzes hydrolysis not only of proteins but of ordinary amides and esters, and much has been learned by use of these simpler substrates. Compounds modeled after portions of the chymotrypsin molecule have been made, and their catalytic effects measured.

• To begin with, it seems very likely that chymotrypsin acts in two stages. In the first stage, acting as an alcohol, it breaks the peptide chain. We recognize this as alcoholics of a substituted amide: nucleophilic acyl substitution. The products are an amine the liberated portion of the substrate molecule and, as we shall

• see shortly, an ester of the enzyme. In the second stage, the enzyme ester is hydrolyzed. This yields a carboxylic acid the other portion of the substrate molecule and the regenerated enzyme, ready to go to work again.

• see shortly, an ester of the enzyme. In the second stage, the enzyme ester is hydrolyzed. This yields a carboxylic acid the other portion of the substrate molecule and the regenerated enzyme, ready to go to work again.

• Now, turning once more to we find that at the reactive site in the enzyme there is a pocket; this pocket is lined with hydrophobic substituents to receive the non-polar group of the substrate and thus hold the molecule in position for hydrolysis. It is the size of this pocket and the nature of its lining that gives the enzyme its specificity; here we find, in a very real sense, Emil Fischer's lock into which the substrate key must fit.

• All these factors are important and can be shown independently to speed up the rate of reactions, and very powerfully, too but they do not seem to be nearly enough to account for the enormous activity of enzymes. Perhaps other factors are involved. It has been suggested, for example, that the pocket in which occurs fits the transition state better than it fits the reactants, so that relief of strain or an increase in van der Waals attractions provides a driving force*. Perhaps the correct factors are being considered but, in extrapolation to enzyme systems, their power has been underestimated.

The source of biological energy. The role of ATP

• In petroleum we have a fuel reserve on which we can draw for energy as long as it lasts. We burn it, and either use the heat produced directly to warm ourselves or convert it into other kinds of energy: mechanical energy to move things about; electrical energy, which is itself transformed at a more convenient place than where the original burning happened into light, or mechanical energy, or back into heat.

• In the same way, the energy our bodies need to keep warm, move about, and build new tissue comes from a food reserve: carbohydrates, chiefly in the form of starch. (We eat other animals, too, but ultimately the chain goes back to a carbohydrate-eater.) In the final analysis, we get energy from food just as we do from petroleum: we oxidize it to carbon dioxide and water.

• This food reserve is not, however, a limited one that we steadily deplete. Our store of carbohydrates and the oxygen to go with it is constantly replenished by the recombining, in plants, of carbon dioxide and water. The energy for recombination comes, of course, from the sun.

• We speak of both petroleum and carbohydrates as sources of energy; we could speak of them as "energy-rich molecules." But the oxygen that is also consumed in oxidation is equally a source of energy. What we really mean is that the energy content of carbohydrates (or petroleum) plus oxygen is greater than that of carbon dioxide plus water. (In total, the bonds that are to be broken are weaker contain more energy than the bonds that are to be formed.) These reactants are, of course, energy-rich only in relation to the particular products we want to convert them into. But this is quite sufficient; in our particular kind of world, these are our sources of entry

• The body takes in carbohydrates and oxygen, then, and eventually gives off carbon dioxide and water. In the process considerable energy is generated. But in what form? And how is it used to move muscles, transport solutes, and build new molecules? Certainly, each of our cells does not contain a tiny fire in which carbohydrates burn merrily, running a tiny steam engine and over which a tiny organic chemist stews up his reaction mixtures. Nor do we contain a central power plant where, again, carbohydrates are burned, and the energy sent about in little steam pipes or electric cables to run muscle-machines and protein-and-fat factories.

• In a living organism, virtually the whole energy system is a chemical one. Energy is generated, transported, and consumed by way of chemical reactions and chemical compounds. Instead of a single reaction with a long plunge from the energy level of carbohydrates and oxygen to that of carbon dioxide and water as in the burning of a log, say there are long series of chemical reactions in which the energy level descends in gentle cascades. Energy resides, ultimately, in the molecules involved; as they move through the organism, they carry energy with them.

•  is nothing magical about this. ATP does not carry about a little bag of energy which it sprinkles on molecules to make them react. Nor does it undergo hydrolysis alongside other molecules and in some mystical way make this energy available to them. ATP simply undergoes reactions only one reaction, really. It phosphorylates, that is, transfers a phosphoryl group, ~~PO3 H2, to some other molecule. For example:

• ATP + R-OH > ADP + R-OP03 H2 Adenosine an Adenosine A phosphate

• ATP is called a "high-energy phosphate" compound, but this simply means that it is a fairly reactive phosphorylating agent. It is exactly as though we were to call acetic anhydride "high-energy acetate" because it is a better acetylating agent than acetic acid. And, indeed, there is a true parallel here: ATP is an anhydride, too, an anhydride of a substituted phosphoric acid, and it is a good phosphorylating agent for much the same reasons that acetic anhydride is a good acetylating agent.

• When ATP loses a phosphoryl group to another molecule, it is converted into ADP, adenosine diphosphate. If ATP is to be regenerated, ADP must itself be phosphorylated, and it is: by certain other compounds that are good enough phosphorylating agents to do this. The important thing in all this is not really the energy level of these various phosphorylating agents so long as they are reactive enough to do the job they must but the fact that the energy level of the carbohydrates and their oxidation products is gradually sinking to the level of carbon dioxide and water. These compounds and oxygen are where the energy is, and ATP is simply a chemical reagent that helps to make it available.

• We have seen that very often factors that stabilize products also stabilize the transition state leading to those products, that is, that often there is a parallel between A/f and act To that extent, the energy level of the various phosphorylating agents may enter in, too: less stable phosphorylating agents less stable, let us say, relative to phosphate anion may in general tend to transfer phosphate to more stable phosphorylating agents. In addition, of course, if any of the phosphate transfers should be too highly endothermic, this would require a prohibitively high aet for reaction.

Biological oxidation of carbohydrates

• Next, let us take a look at the overall picture of the biological oxidation of carbohydrates. We start with glycogen ("sugar-former"), the form in which carbohydrates are stored in the animal body. This, we have seen (Sec. 35.9), is a starch-like polymer of D-glucose.

• The trip from glycogen to carbon dioxide and water is a long one. It is made up of dozens of reactions, each of which is catalyzed by its own enzyme system. Each of these reactions must, in turn, take place in several steps, most of them unknown. (Consider what is involved in the "reaction" catalyzed by chymotrypsin.) We can divide the trip into three stages, (a) First, glycogen is broken down into its component D-glucose molecules, (b) Then, in glycolysis ("sugar-splitting"), D-glucose is itself broken down, into three-carbon compounds, (c) These, in respiration, are converted into carbon dioxide and water. Oxygen appears in only the third stage; the first two are anaerobic ("without-air") processes.

• The first stage, cleavage of glycogen, is simply the hydrolytic cleavage of acetal linkages (Sec. 34.16), this time enzyme catalyzed.

• No oxygen is consumed, and we move only a little way down the energy hill toward carbon dioxide and water. What is important is that a start has been made in breaking the five carbon-carbon bonds of glucose, and that two molecules of ADP are converted into ATP. (ATP is required for some of the steps of glycolysis, but there is a net production of two molecules of ATP for each molecule of glucose consumed.)

• The third stage, respiration, is a complex system of reactions in which molecules provided by glycolysis are oxidized. Oxygen is consumed, carbon dioxide and water are formed, and energy is produced.

• Let us look at the linking-up between glycolysis and respiration. Ordinarily, the energy needs of working muscles are met by respiration. But, during short periods of vigorous exercise, the blood cannot supply oxygen enough for respiration to carry the entire load; when this happens, glycolysis is called upon to sup* ply the energy difference. The end-product of glycolysis, lactic acid, collects in the muscle, and the muscle feels tired. The lactic acid is removed by the blood and rebuilt into glycogen, which is ready for glycolysis.

• The last step of glycolysis is reduction of pyruvic acid to lactic acid. (The reducing agent is, incidentally, an old acquaintance, reduced nicotinamide adenine 

•  It is as acetyl CoA that the products of glycolysis are fed into the respiration cycle. The acetyl CoA that is fuel for respiration comes not only from carbohydrates but also from the breakdown of amino acids and fats. It is thus the common link between all three kinds of food and the energy-producing process. (Acetyl CoA is even more than that: as we shall see, it is the building block from which the long chains of fatty acids are synthesized.)

• Thiols are sulfur analogs of alcohols. They contain the sulfhydryl group, SH, which plays many parts in the chemistry of biomolecules. Easily oxidized, two SH groups are converted into disulfide links, S-S-, which hold together different peptide chains or different parts of the same chain. (See, for example, oxytocin on p. 1 143.) Thiols form the same kinds of derivatives as alcohols: /A/anthers, /A/0acetals, thiol esters. Thiol ester groups show the chemical behavior \\e would expect they undergo nucleophilic acyl substitution, and they make a-hydrogens acidic this last more effectively than their oxygen counterparts.

Mechanism of a biological oxidation

• Now let us take just one of the many steps in carbohydrate oxidation and look at it in some detail Although there is no net oxidation in glycolysis, certain individual reactions do involve oxidation and reduction. About mid-way in the eleven steps, H2O3P-~O-CH2CHOHCHO H2O3P O CH2CHOHCOOH D-Glyceraldehyde-3-phosphate 3-Phosphoglyceric acid at D-glyceraldehyde-3-phosphate and its oxidation to 3-phosphoglyceric acid. In the course of this conversion, a phosphate ion becomes attached to ADP to generate a molecule of ATP.Two reactions are actually involved. First, D-glyceraldehyde-3-phosphate is oxidized, but not directly to the corresponding acid, 3-phosphog1yceric acid.

 

• The NADH produced is also available to do its job, that of reducing agent. It may, for example, reduce pyruvate to lactate in the last step of glycolysis. The extra electrons that make it a reducing agent are passed along, and ultimately are accepted by molecular oxgang.

• We are in a strange, complex chemical environment here, but in it we recognize familiar kinds of compounds hemiacetals, esters, anhydrides, carboxylic acids and familiar kinds of reactions nucleophilic carbonyl addition, hydride transfer, nucleophilic acyl substitution.

Biosynthesis of fatty acid

• When an animal eats more carbohydrate than it uses up, it stores the excess: some as the polysaccharide glycogen (Sec. 35.9), but most of it as fats. Fats, we know (Sec. 33.2), are triacylglycerols, esters derived (in most cases) from long straight-chain carboxylic acids containing an even number of carbon atoms. These even numbers, we said, are a natural consequence of the way fats are synthesized in biological systems. 

• There are even numbers of carbons in fatty acids because the acids are built up, two carbons at a time, from acetic acid units. These units come from acetyl CoA: the thiol ester derived from acetic acid and coenzyme A (Sec. 37.4). The acetyl CoA itself is formed either in glycolysis, as we have seen, or by oxidation of fatty .

• Let us see how fatty acids are formed from acetyl CoA units. As before, we must realize that every reaction is catalyzed by specific enzyme and proceeds by several steps steps that in some direct, honest-to-goodness chemical way, involved the enzyme.

• First, acetyl CoA takes up carbon dioxide (1) to form malonyl CoA. (To illustrate the point made above: this does not happen directly; carbon dioxide combines.

• Enzymes are marvelous catalysts. Yet, even with their powerful help, these biological reactions seek the easiest path. In doing this, they take advantage of the same structural effects that the organic chemist does: the acidity of a-hydrogens, the leaving ability of a particular group, the ease of decarboxylation of j8-keto acids.

Nucleoproteins and nucleic acids

• In every living cell there are found nucleoproteins: substances made up of proteins combined with natural polymers of another kind, the nucleic acids. Of all fields of chemistry, the study of the nucleic acids is perhaps the most exciting, for these compounds are the substance of heredity. Let us look very briefly at the structure of nucleic acids and then, in the next section, see how this structure may be related to their literally vital role in heredity.

• Although chemically quite different, nucleic acids resemble proteins in a fundamental way: there is a long chain a backbone that is the same (except for length) in all nucleic acid molecules; and attached to this backbone are various groups, which by their nature and sequence characterize each individual nucleic acid.

• Where the backbone of the protein molecule is a polyamide chain (a polypeptide chain), the backbone of the nucleic acid molecule is a polyester chain (called a polynucleotide chain). The ester is derived from phosphoric acid (the acid portion) and a sugar (the alcohol portion).

• acids (RNA), and D-2-deoxyribose in the group known as deoxyribonucleic acids (DNA), (The prefix 2-deoxy simply indicates the lack of an OH group at the 2-position.) The sugar units are in the furanose form, and are joined to phosphate through the C-3 and C-5 hydroxyl groups.

• The proportions of these bases and the sequence in which they follow each other along the polynucleotide chain differ from one kind of nucleic acid to another. This primary structure is studied in essentially the same way as the structure of proteins: by hydrolytic degradation and identification of the fragments. In this way, and after seven years of work, Robert W. Holley and his collaborators at Cornell University, determined the exact sequence of the 77 nucleotides in the molecule of one kind of transport RNA (p. 1181).

• What can we say about the secondary structure* of nucleic acids? The following picture of DNA fits both chemical and x-ray evidence. Two polynucleotide chains, identical but heading in opposite directions, are wound about each other to form a double helix 18 A in diameter (shown schematically in). Both helixes are right-handed and have ten nucleotide residues per turn.

• So far we have discussed only the nucleic acid portion of nucleoproteins. There is evidence that in one nucleoprotein (found in fish sperm), a polyarginine chain lies in one of the grooves of the double helix, held by electrostatic forces between the negative phosphate groups of the polynucleotide (which face the outside of the helix) and the positive guanidium groups of the arginine residues.

• So far we have discussed only the nucleic acid portion of nucleoproteins. 

Chemistry and heredity. The genetic code

• Just how is the structure of nucleic acids related to their function in heredity? Nucleic acids control heredity on the molecular level. The double helix of DNA is the repository of the hereditary information of the organism. The information is stored as the sequence of bases along the polynucleotide chain; it is a message "written" in a language that has only four letters, A, G, T, C (adenine, guanine, thymine, cytosine).

• DNA must both preserve this information and use it. It does these things through two properties: (a) DNA molecules can duplicate themselves, that is, can bring about the synthesis of other DNA molecules identical with the originals; and (b) DNA molecules can control the synthesis, in an exact and specific way, of the proteins that are characteristic of each kind of organism.

• First, there is the matter of self-duplication. The sequence of bases in one chain of the double helix controls the sequence in the other chain. The two chains fit together (as F. H. C. Crick of Cambridge University puts it) like a hand and a glove. They separate, and about the hand is formed a new glove, and inside the glove is formed a new hand. Thus, the pattern is preserved, to be handed down to the next generation.

• Next, there is the matter of guiding the synthesis of proteins. A particular sequence of bases along a polynucleotide chain leads to a particular sequence of amino acid residues along a polypeptide chain. A protein has been likened to a long sentence written in a language of 20 letters: the 20 different amino acid residues. But the hereditary message is written in a language of only four letters; it is written in a code, with each word standing for a particular amino acid.

• The genetic code has been broken, but research continues, aimed at tracking down the lines of communication. DNA serves as a template on which molecules of. RNA are formed. It has been suggested that the double helix of DNA partially Um foils, and about the individual strands are formed chains of RNA; the process thus resembles self-duplication of DNA, except that these new chains contain.

• A difference of a single base in the DNA molecule, or a single error in the "reading" of the code can cause a change in the amino acid sequence. The tiny defect in the hemoglobin molecule that results in sickle-cell anemia (p. 1152) has been traced to a single gene a segment of the DNA chain where, perhaps, the codon GUG appears instead of GAG. There is evidence that antibiotics, by altering the ribosome, cause misreading of the code and death to the organism.

• Thus, the structure of nucleic acid molecules determines the structure of protein molecules. The structure of protein molecules, we have seen, determines the way in which they control living processes. Biology is becoming more and more a matter of shapes and sizes of molecules.

• At the beginning of this book, we said that the structural theory is the basis of the science of organic chemistry. It is much more than that: the structural theory is the basis of our understanding of life.