1.

INTRODUCTION TO BIOCHEMISTRY Introduction Biochemistry can be defined as the science concerned with the chemical basis of life (bios life). The cell is the structural unit of living systems. Thus, biochemistry can also be described as the science concerned with the chemical constituents of living cells and with the reactions and processes they undergo. By this definition, biochemistry encompasses large areas of cell biology, of molecular biology, and of molecular genetics. Fifteen to twenty billion years ago, the universe arose as a cataclysmic eruption of hot, energyrich subatomic particles. Within seconds, the simplest elements (hydrogen and helium) were formed. As the universe expanded and cooled, material condensed under the influence of gravity to form stars. Some stars became enormous and then exploded as supernovae, releasing the energy needed to fuse simpler atomic nuclei into the more complex elements. Thus were produced, over billions of years, the Earth itself and the chemical elements found on the Earth today. About four billion years ago, life arosesimple microorganisms with the ability to extract energy from organic compounds or from sunlight, which they used to make a vast array of more complex biomolecules from the simple elements and compounds on the Earths surface. Biochemistry asks how the remarkable properties of living organisms arise from the thousands of different lifeless biomolecules. When these molecules are isolated and examined individually, they conform to all the physical and chemical laws that describe the behavior of inanimate matteras do all the processes occurring in living organisms. The study of biochemistry shows how the collections of inanimate molecules that constitute living organisms interact to maintain and perpetuate life animated solely by the physical and chemical laws that govern the nonliving universe. The most obvious quality of living organisms is that they are complicated and highly organized. For example, organisms large enough to be seen with the naked eye are composed of many cells, typically of many types. In turn, these cells possess subcellular structures or organelles, which are complex assemblies of very large polymeric molecules or macromolecules. These macromolecules themselves show an exquisite degree of organization in their intricate threedimensional architecture, even though they are composed of simple sets of chemical building blocks, such as sugars and amino acids. Indeed, the complex three-dimensional structure of a macromolecule, known as its conformation, is a consequence of interactions between the monomeric units, according to their individual chemical properties. Biomolecules Biomolecules deals with the organic molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products.

Biomolecules are the building blocks of life and perform important functions in living organisms. It is a long jump from simple biomolecules to cellular structures that can be seen with the light microscope. Figure 1.1 illustrates the structural hierarchy in cellular organization. The monomeric subunits in proteins, nucleic acids, and polysaccharides are joined by covalent bonds. In supramolecular complexes, however, macromolecules are held together by noncovalent interactionsmuch weaker, individually, than covalent bonds. Among these noncovalent interactions are hydrogen bonds (between polar groups), ionic interactions (between charged groups), hydrophobic interactions (among nonpolar groups in aqueous solution), and van der Waals interactionsall of which have energies substantially smaller than those of covalent bonds. The large numbers of weak interactions between macromolecules in supramolecular complexes stabilize these assemblies, producing their unique structures.

FIGURE 1.1 Structural hierarchy in the molecular organization of cells. In this plant cell, the nucleus is an organelle containing several types of supramolecular complexes, including chromosomes. Chromosomes consist of macromolecules of DNA and many different proteins. Each type of macromolecule is made up of simple subunits DNA of nucleotides (deoxyribonucleotides), for example.

Metabolites and Macromolecules The major precursors for the formation of biomolecules are water, carbon dioxide, and three inorganic nitrogen compoundsammonium (NH4+), nitrate (NO3-), and dinitrogen (N2). Metabolic processes assimilate and transform these inorganic precursors through ever more complex levels of biomolecular order. In the first step, precursors are converted to metabolites,

simple organic compounds that are intermediates in cellular energy transformation and in the biosynthesis of various sets of building blocks: amino acids, sugars, nucleotides, fatty acids, and glycerol. By covalent linkage of these building blocks, the macromolecules are constructed: proteins, polysaccharides, polynucleotides (DNA and RNA), and lipids. (Strictly speaking, lipids contain relatively few building blocks and are therefore not really polymeric like other macromolecules; however, lipids are important contributors to higher levels of complexity.) Interactions among macromolecules lead to the next level of structural organization, supramolecular complexes. Here, various members of one or more of the classes of macromolecules come together to form specific assemblies serving important subcellular functions. Examples of these supramolecular assemblies are multifunctional enzyme complexes, ribosomes, chromosomes, and cytoskeletal elements. For example, a eukaryotic ribosome contains four different RNA molecules and at least 70 unique proteins. These supramolecular assemblies are an interesting contrast to their components because their structural integrity is maintained by noncovalent forces, not by covalent bonds. These noncovalent forces include hydrogen bonds, ionic attractions, van der Waals forces, and hydrophobic interactions between macromolecules. Such forces maintain these supramolecular assemblies in a highly ordered functional state. Although noncovalent forces are weak (less than 40 kJ/mol), they are numerous in these assemblies and thus can collectively maintain the essential architecture of the supramolecular complex under conditions of temperature, pH, and ionic strength that are consistent with cell life.

Chemical Foundations of life Hydrogen, oxygen, carbon, and nitrogen constitute more than 99% of the atoms in the human body, with most of the H and O occurring as H2O. What property unites H, O, C, and N and renders these atoms so suitable to the chemistry of life? It is their ability to form covalent bonds by electron-pair sharing. Furthermore, H, C, N, and O are among the lightest elements of the periodic table capable of forming such bonds. Because the strength of covalent bonds is inversely proportional to the atomic weights of the atoms involved, H, C, N, and O form the strongest covalent bonds. Two other covalent bond-forming elements, phosphorus (as phosphate -OPO3-2 derivatives) and sulfur also play important roles in biomolecules. The trace elements represent a miniscule fraction of the weight of the human body, but all are essential to life, usually because they are essential to the function of specific proteins, including enzymes. The oxygen-transporting capacity of the hemoglobin molecule, for example, is absolutely dependent on four iron ions that make up only 0.3% of its mass. Biomolecules Are Compounds of Carbon with a Variety of Functional Groups The chemistry of living organisms is organized around carbon, which accounts for more than half the dry weight of cells. Carbon can form single bonds with hydrogen atoms, and both single

and double bonds with oxygen and nitrogen atoms (Fig. 1.2). Of greatest significance in biology is the ability of carbon atoms to form very stable carboncarbon single bonds. Each carbon atom can form single bonds with up to four other carbon atoms. Two carbon atoms also can share two (or three) electron pairs, thus forming double (or triple) bonds.

Figure 1.2. Versatility of carbon bonding. Carbon can form covalentsingle, double, and triple bonds (in red), particularly with other carbon atoms. Triple bonds are rare in biomolecules.

The four single bonds that can be formed by a carbon atom are arranged tetrahedrally, with an angle of about 109.5 between any two bonds (Fig. 1.3) and an average length of 0.154 nm. There is free rotation around each single bond, unless very large or highly charged groups are attached to both carbon atoms, in which case rotation may be restricted. A double bond is shorter (about 0.134 nm) and rigid and allows little rotation about its axis.

Figure 1.3. Geometry of carbon bonding. (a) Carbon atoms have a characteristic tetrahedral arrangement of their four single bonds. (b) Carboncarbon single bonds have freedom of rotation, as shown for the compound ethane (CH3OCH3). (c) Double bonds are shorter and do not allow free rotation. The two doubly bonded carbons and the atoms designated A, B, X, and Y all lie in the same rigid plane.

Covalently linked carbon atoms in biomolecules can form linear chains, branched chains, and cyclic structures. To these carbon skeletons are added groups of other atoms, called functional groups, which confer specific chemical properties on the molecule. It seems likely that the bonding versatility of carbon was a major factor in the selection of carbon compounds for the molecular machinery of cells during the origin and evolution of living organisms. No other chemical element can form molecules of such widely different sizes and shapes or with such a variety of functional groups. Most biomolecules can be regarded as derivatives of hydrocarbons, with hydrogen atoms replaced by a variety of functional groups to yield different families of organic compounds. Typical of these are alcohols, which have one or more hydroxyl groups; amines, with amino groups; aldehydes and ketones, with carbonyl groups; and carboxylic acids, with carboxyl groups (Fig. 1.4). Many biomolecules are polyfunctional, containing two or more different kinds of functional groups (Fig. 1.5), each with its own chemical characteristics and reactions. The chemical personality of a compound is determined by the chemistry of its functional groups and their disposition in three-dimensional space.

Figure 1.4. Some common functional groups of biomolecules. In this figure we use R to represent any substituent. It may be as simple as a hydrogen atom, but typically it is a carbon-containing moiety. When two or more substituents are shown in a molecule, we designate them R1, R2, and so forth.

Figure 1.5. Several common functional groups in a single biomolecule. Acetyl-coenzyme A (often abbreviated as acetyl-CoA) is a carrier of acetyl groups in some enzymatic reactions.

The Unique Properties of Carbon Living matter consists of a relatively small number of elements: C, H, O, N, P and S, all of which readily form covalent bonds. The predominance of carbon in living matter is no doubt a result of its tremendous chemical versatility compares with all other elements. Carbon has the unique ability to form a virtually infinite number of compounds as a result of its capacity to make as many as four highly stable covalent bonds (including single, double and triple bonds) combined with its ability to form covalently linked C-C chains of unlimited extent. Thus, of the over 17 million chemical compounds that are presently known, nearly 90% are organic (carbon-contain) substances. Let us examine the other elements in the periodic table to ascertain why they lack these combined properties. Only five elements B, C, N, Si and P, have the capacity to make three or more bonds each and thus to perform chains of covalently linked atoms that can also have pendant side chains. The other elements are either metals, which tend to form ions rather than covalent bonds; noble gasses which are chemically inert; or atoms such as H or O that can each make only one or two covalent bonds. However, although B, N Si and P can each participate in at least three covalent bonds, they are for reasons indicated below, unsuitable as the basis of a complex chemistry. Boron, having fewer valence electrons (3) than valence orbitals (4), is electron deficient. This severely limits the types and stabilities of compounds that B can form. Nitrogen has the opposite problem: its 5 valence electrons make it electron rich. The repulsion between the lone pairs of electrons on covalently bonded N atoms serve to greatly reduce the bond energy of N-N bond (171 kJmol-1 vs 348 kJmol-1 for a C-C single bond) relative to the unusually stable triple bond of the N2 molecule (946 kJmol-1). Silicon and carbon being in the same column of the periodic table might be expected to have similar chemistries. Silicons large atomic radius, however, prevents two Si atoms from approaching each other closely enough to gain much orbital overlap. Consequently Si-Si bonds are weak (177 kJmol-1) and the corresponding multiple bonds are

rarely stable. Phosphorus being below N in the periodic table, forms even less stable chains of covalently bonded atoms. The foregoing does not imply that heteronuclear bonds are unstable. On the contrary, proteins contain C-N-C linkages, carbohydrates have C-O-C linkages, and nucleic acids possess C-O-PO-C linkages. However, these heteronuclear linkages are less stable that are C-C bonds. Indeed, they usually form sites of chemical cleavage in the degradation of macromolecules and, conversely are the bonds formed when monomer units are linked together to form macromolecules. In the same vain, homonuclear linkages other than C-C bonds are so reactive that they are, with the exception of S-S bonds in proteins, extremely rare in biological systems. Three-Dimensional Structure Is Described by Configuration and Conformation The covalent bonds and functional groups of a biomolecule are, of course, central to its function, but so also is the arrangement of the molecules constituent atoms in three-dimensional space its stereochemistry. A carbon-containing compound commonly exists as stereoisomers, molecules with the same chemical bonds but different stereochemistrythat is, different configuration, the fixed spatial arrangement of atoms. Interactions between biomolecules are invariably stereospecific, requiring specific stereochemistry in the interacting molecules. Configuration is conferred by the presence of either (1) double bonds, around which there is no freedom of rotation, or (2) chiral centers, around which substituent groups are arranged in a specific sequence. The identifying characteristic of configurational isomers is that they cannot be interconverted without temporarily breaking one or more covalent bonds. Figure 1.6 shows the configurations of maleic acid and its isomer, fumaric acid. These compounds are geometric, or cis-trans, isomers; they differ in the arrangement of their substituent groups with respect to the nonrotating double bond (Latin cis, on this sidegroups on the same side of the double bond; trans, acrossgroups on opposite sides). Maleic acid is the cis isomer and fumaric acid the trans isomer; each is a well-defined compound that can be separated from the other, and each has its own unique chemical properties. A binding site (on an enzyme, for example) that is complementary to one of these molecules would not be a suitable binding site for the other, which explains why the two compounds have distinct biological roles despite their similar chemistry.

Figure 1.6. Configurations of geometric isomers. Isomers such as maleic acid and fumaric acid cannot be interconverted without breaking covalent bonds, which requires the input of much energy.

In the second type of configurational isomer, four different substituents bonded to a tetrahedral carbon atom may be arranged two different ways in spacethat is, have two configurations (Fig. 1.7)yielding two stereoisomers with similar or identical chemical properties but differing in certain physical and biological properties. A carbon atom with four different substituents is said to be asymmetric, and asymmetric carbons are called chiral centers (Greek chiros, hand; some stereoisomers are related structurally as the right hand is to the left). A molecule with only one chiral carbon can have two stereoisomers; when two or more (n) chiral carbons are present, there can be 2n stereoisomers. Some stereoisomers are mirror images of each other; they are called enantiomers (Fig. 1.7). Pairs of stereoisomers that are not mirror images of each other or those which differ at only one of the asymmetric centers are called diastereomers (Fig. 1.8).
FIGURE 1.7. Molecular asymmetry: chiral and achiral molecules. (a) When a carbon atom has four different substituent groups (A, B, X, Y), they can be arranged in two ways that represent nonsuperimposable mirror images of each other (enantiomers). This asymmetric carbon atom is called a chiral atom or chiral center. (b) When a tetrahedral carbon has only three dissimilar groups (i.e., the same group occurs twice), only one configuration is possible and the molecule is symmetric, or achiral. In this case the molecule is superimposable on its mirror image: the molecule on the left can be rotated counterclockwise (when looking down the vertical bond from A to C) to create the molecule in the mirror.

Figure 1.8. Two types of stereoisomers. There are four different 2,3-disubstituted butanes (n _ 2 asymmetric carbons, hence 2n=4 stereoisomers). Each is shown in a box as a perspective formula and a ball-and-stick model, which has been rotated to allow the reader to view all the groups. Some pairs of stereoisomers are mirror images of each other, or enantiomers. Other pairs are not mirror images; these are diastereomers.

Nomenclature of stereoisomers Given the importance of stereochemistry in reactions between biomolecules, biochemists must name and represent the structure of each biomolecule so that its stereochemistry is unambiguous. For naming there are two systems: D, L and R, S system. D, L system As Louis Pasteur first observed, enantiomers have nearly identical chemical properties but differ in a characteristic physical property, their interaction with plane-polarized light. Enantiomeric molecules display a property called optical activity-the ability to rotate the plane of polarization. In separate solutions, two enantiomers rotate the plane of plane-polarized light in opposite directions, but an equimolar solution of the two enantiomers (a racemic mixture) shows no optical rotation. Compounds without chiral centers do not rotate the plane of plane-polarized light. Clockwise rotation of incident light is referred to as dextrorotatory behavior and counterclockwise is called levorotatory behavior. Therefore, the enantiomer that rotates the light clockwise is represented with D (for dextrorotatory) or (+) whereas one that rotates the light counterclockwise is shown as L (for levorotatory) or (-).In the D, L system of nomenclature, the (+) and (-) isomers of glyceraldehyde are denoted as D-glyceraldehyde and L-glyceraldehyde, respectively (Figure 1.9).

Figure 1.9. D and L isomers of glyceraldehyde and serine

In spite of its widespread acceptance, problems exist with the D,L system of nomenclature. For example, this system can be ambiguous for molecules with two or more chiral centers. To address such problems, the (R,S ) system of nomenclature for chiral molecules was proposed in 1956 by Robert Cahn, Sir Christopher Ingold, and Vladimir Prelog. R, S system In this more versatile system, priorities are assigned to each of the groups attached to a chiral center on the basis of atomic number, atoms with higher atomic numbers having higher priorities. For naming in the RS system, the chiral atom is viewed with the group of lowest priority (4 in the figure 1.10) pointing away from the viewer. If the priority of the other three groups (1 to 3) decreases in clockwise order, the configuration is (R) (Latin rectus, right); if in counterclockwise order, the configuration is (S) (Latin sinister, left). In this way each chiral carbon is designated either (R) or (S), and the inclusion of these designations in the name of the compound provides an unambiguous description of the stereochemistry at each chiral center.
Figure 1.10. Priorities assigned to each of the groups around chiral center

If two of the atoms coordinated to a chiral center are identical, the atoms bound to these two are considered for priorities. For such purposes, the priorities of certain functional groups found in the molecules are in the following order:

Fischer Projection A representation of the absolute configuration of an asymmetrical C-atom, especially the carbons of a sugar, in which the substituents further from the observer are shown above and below and the substituents closer to the observer are shown to the right and left. OR Representation of a 3D molecule as a flat structure where a tetrahedral carbon is represented as two crossed lines

Manipulation of Fischer Projections: 1. Fischer Projection can be rotated by 180 only

Rotation by 90 or -90 (270 ) invert the stereochemistry

2. If one group of the Fischer projection is held steady, the other groups can be rotated either clockwise or counter clockwise.

Assigning R and S-configurations to Fischer projections 1. Assign priorities to the four substituents according to the Cahn-Ingold-Prelog convention. 2. Perform the two allowed manipulations of the Fischer projection to place the lowest priority group on one of the vertical positions (either top or bottom) 3. If the priorities of the other three groups (1-2-3) proceed clockwise, the stereogenic center is assigned as R. If the priorities of the other three groups (1-2-3) proceed counter clockwise, the stereogenic center is assigned as S.