Introduction to Biological Chemistry

Biological Chemistry by Wayne Best - Lecture 1

Once upon a time it was considered impossible to make organic compounds from inorganic ones in vitro. It was believed only living things could take inorganic nutrients and convert them into organic molecules, and that such transformations required some, as yet unidentified, magical life force. It was not until 1828, when a German chemist, Friedrich Whler, synthesised urea that this theory, called vitalism, began to lose favour. It was finally discredited in 1844 when another German chemist, Adolf Klbe, synthesised acetic acid from inorganic materials. Today, we take it for granted that there is nothing magical about living systems. Nevertheless, most of the chemical reactions which occur readily in vivo are quite remarkable and a chemist would find it very difficult to mimic them in the laboratory, even using reactive reagents and extreme temperatures. How then do biological systems achieve these difficult transformations in an aqueous environment at essentially neutral pH and ambient temperature...? The answer is of course, highly specific and efficient catalysts called enzymes. Enzymes belong to that very important class of compound called proteins. Although all enzymes are proteins, not all proteins are enzymes. There are several classes of proteins, each performing a vital role within living systems depending on their structures. Proteins such as keratin (nails and hair), collagen (tendons) and myosin (muscle) provide structural support and are usually fibrous in nature and insoluble in water. Transport proteins such as haemoglobin (for the transport of oxygen in blood) and regulatory proteins such as the hormone insulin are globular in structure and soluble in water. Similarly, the catalytic proteins (enzymes) are generally globular in structure and water soluble.
R H2N O OH R R OH O H2N O H N R O OH H2N O

Proteins are polymers consisting of amino acid subunits (Figure 1). Although there are over 300 naturally occurring amino acids only about 20 of them are found in proteins and, not surprisingly, these are referred to as protein amino acids (Table 1). Note that all the -amino acids, except for glycine, are chiral, and that consequently proteins and enzymes are also chiral. Protein amino acids are L, which means that they have the S configuration (except for cysteine which is R). These protein amino acids are often divided into two groups referred to as essential or nonessential. These terms are somewhat misleading since they refer only to whether the amino acids can be synthesised by the body (nonessential) or whether they must be obtained from dietary sources (essential). Of course both these groups are essential for the synthesis of proteins. Of special significance amongst the amino acids is cystine, the disulphide of cysteine. These disulphide linkages are of considerable importance in determining the structure of proteins. While large polymers of amino acids are referred to as proteins, smaller oligomers are usually called polypeptides, and where there are less than 10 amino acid residues the terms dipeptide, tripeptide, etc are usually used. The amide bonds connecting adjacent amino acids are often referred to as peptide bonds. There are numerous methods for forming these peptide bonds in the laboratory. Ironically, their formation in biological systems is catalysed by enzymes, themselves polypeptides. Amide (peptide) bonds have a large contribution from the zwitterionic resonance structure which imparts a degree of double bond character to the bond (around 50%). This means that there is restricted rotation about the bond and all six atoms lie in one
R H N R O N H O R OH proteins

H2N

-amino acid

-amino acid

dipeptide

tripeptide

Figure 1. From amino acids to proteins.

plane. This higher bond order is also reflected in the bond angles and distances (Figure 2) and has important implications for the conformation peptides can adopt.
1.24 O N 1.53 H 1.47 1.32 O N H
+

Table 1. Protein amino acids

CO2H NH2

Glycine (Gly - G) Alanine (Ala - A) Valine (Val - V)*

CO2H NH2

CO2H NH2
CO2H NH2

Leucine (Leu - L)* Isoleucine (Ile - I)*

Figure 2. The peptide bond

CO2H NH2

The structure of a protein is divided into four categories, given the familiar terms, primary, secondary, tertiary and quaternary. Primary structure refers to the order of amino acids in the polypeptide chain and the location of disulphide bonds if any are present, ie it describes the covalent bonds in the protein. When drawing primary structures of polypeptides the convention is to place the free amino group on the left and the free carboxyl group on the right. While this may seem unimportant for normal chemical structures it becomes crucial for the shorthand representations, where reversing the order gives rise to a different molecule. The single letter abbreviations are normally only used for large polypeptides and proteins. Secondary structure refers to the three dimensional relationships of amino acids which are close together in the primary sense. There are several common secondary structural types, the most important of which are the -helix (Figure 3), stabilised by intramolecular hydrogen bonds, and the -pleated sheet (Figure 4), stabilised by either intermolecular or remote hydrogen bonds. The -helix can be either left or right handed with the right handed helix the more stable and usual form. There are about 3.6 amino acid residues per turn of the helix and the gap between the turns is approximately 5.4 Angstroms. This allows for the maximum hydrogen bonding between the carbonyls and the amide hydrogens. If you were to look down the axis of one of these helices you would see that there is virtually no free space in the interior and all the substituents on the carbons radiate outwards. The -pleated sheet can be either parallel, where all the N-termini

CO2H S NH2
CO2H HS NH2

Methionine (Met - M)* Cysteine (Cys - C) Serine (Ser - S) Threonine (Thr - T)*

CO2H HO NH2

CO2H HO NH2

CO2H H2N
H2N H N NH
O HO O H2 N CO2H NH2 CO2H NH2

NH2
CO2H NH2

Lysine (Lys - K)* Arginine (Arg - R)

Aspartic acid (Asp - D) Asparagine (Asn - N) Glutamic acid (Glu - E)

CO2H HO O CO2H H2N O NH2 NH2

Glutamine (Gln - Q)

N N H

CO2H NH2
CO2H NH2

Histidine (His - H)

Phenylalanine (Phe - F)*

HO

CO2H NH2

Tyrosine (Tyr - Y)

H N

CO2H NH2

Tryptophan (Trp - W)*

CO2H NH

Proline (Pro - P)

* essential (dietary requirement)

Figure 4. Antiparallel -pleated sheet

Figure 3. Right handed -helix

are oriented in the same direction, and antiparallel, which has alternate chains running in the opposite direction. Most fibrous proteins such as muscle tissue are made almost exclusively of pleated sheets, whereas most globular proteins contain a number of different regions of secondary structure including both sheets and helices. Tertiary structure refers to the overall three dimensional structure of the entire protein, which usually includes several different regions of secondary structure. Quaternary structure relates to proteins which may exist as dimers, trimers or other oligomeric species. The majority of proteins exist as monomers and therefore have no quaternary structure. The secondary, tertiary and any quaternary structures of proteins are determined entirely by their primary structures. That is, the order of amino acids in the peptide chain determines the way the chain twists and folds back on itself and even whether it forms stable dimers or higher oligomers.

In trying to understand why proteins form these complex yet well defined three dimensional structures, we must consider the environment in which they are formed. At physiological pH (about 7.4) carboxylic acids will be almost completely deprotonated and exist as the carboxylate anion. Similarly, amino groups will be protonated and bear a positive charge. Remember also that it is an aqueous environment which means polar groups will be well solvated but the hydrophobic hydrocarbon residues will not. Now let us re-examine the list of amino acids and we will see that they can be divided into four main groups those with i) non-polar, essentially hydrophobic substituents, eg phenylalanine; ii) negatively charged substituents, eg glutamic acid; iii) positively charged substituents, eg histidine; iv) polar but not charged substituents, eg serine. Because of prolines somewhat unique cyclical nature and secondary amino group it has the action of terminating any -helices. Proteins, like any other molecule, will arrange themselves so as to reduce any unfavourable interactions and to adopt the thermodynamically most stable conformation under the circumstances. For example, the hydrophobic alkyl groups tend to aggregate together towards the interior of the protein away from water, while the polar amino acid residues tend to be found on the exterior surfaces of the protein, solvated by water molecules.

Furthermore, negatively charged residues (eg aspartic acid) would be more stable when located next to a positively charged residue such as arginine rather than another negatively charged residue. Note that the most stable conformation may vary depending on a number of factors, in particular pH. This is one reason why most enzymes only work within a narrow pH range. The relatively weak intramolecular forces which are responsible for secondary, tertiary, and quaternary structure are easily disrupted by heat, changes in pH, and other things which can interfere with hydrogen bonds. This disruption, called denaturation, obviously results in inactivation of the catalytic properties of the enzyme. If the secondary and tertiary structure is destroyed but the primary structure is left intact the protein can often reassemble its original tertiary structure when the correct physiological conditions are restored. Its biological activity is then also restored The majority of amino acid residues in enzymes simply help in a small way to define its tertiary structure. Often they can be replaced with a similar amino acid without any loss in activity. Other amino acids have a more important role in defining the tertiary structure or may be involved in binding to the substrate or coenzymes. Changing these amino acids can have a significant change on the enzyme efficiency and specificity. Only a few amino acids are actually involved in any catalytic processes at the active site. Changing
A d e no s ine trip ho s p hate (A T P )
N O HO P O
-

NH 2 N N

these almost always results in loss of catalytic activity. Many enzymes are not catalytically active in their own right and require what is called a cofactor. These cofactors can be metal ions or relatively small organic molecules such as vitamins, when they are usually referred to as coenzymes. The term coenzyme can be somewhat misleading since unlike enzymes, many coenzymes are not catalytic and are required in stoichiometric amounts. Coenzymes that are required in stoichiometric amounts are often, and more correctly, referred to as cosubstrates. The majority of catalysts used in conventional chemistry contain metals, and in particular transition metals. It will therefore come as no surprise that about 25% of all enzymes contain a tightly bound metal or require a metal cofactor for activity. Since enzymes are catalysts they can only increase the rate of reactions, they can not make energetically unfavourable reactions proceed. If an endothermic reaction is required, it must be provided with energy by coupling it to an energy rich cosubstrate in an exothermic reaction so that the overall transformation is energetically favoured. These exothermic coupling reactions usually come in the form of the hydrolysis of high energy phosphates, the most important of which is the hydrolysis of ATP to give ADP. These high energy phosphates are an example of non catalytic coenzymes. In general, one molecule of ATP is hydrolysed to ADP for every molecule of substrate converted to product.

O O P O
-

O O P O
-

N O O

Table 2. Common phosphate cofactors Phosphate G (kJ/mol) phosphoenolpyruvate -62 carbamoyl phosphate -52 1,3-bisphosphoglycerate -49 creatine phosphate -43 ATP (to ADP) -30 ADP (to AMP) -28 pyrophosphate -28 glucose 1-phosphate -21 fructose 6-phosphate -16 AMP -14 glucose 6-phosphate -14 glycerol 3-phosphate -9

OH

OH

NH 2

A d e no s ine d ip ho s p hate (A D P )
O HO P O
-

N N N

O OH

O O
-

O HO

P O

P O
-

OH

OH

Figure 5. Hydrolysis of ATP to ADP

An important feature of enzymes is that most show high enantioselectivty. Synthetic chemists have taken advantage of mother natures chiral catalysts for many years and there are numerous reports in the literature where they have been used in vitro. For example, oxynitrilase (isolated from almond flour) has been used to convert benzaldehydes into chiral cyanohydrins with high enantiomeric excess (ee). Similarly, pig liver esterase, has often been used to selectively produce a single enantiomer from prochiral diesters (Fiure 6).
O H oxynitrilase KCN / AcOH pH 5.4 H OH CN 99% ee

CO2Me H pig liver esterase pH 8 CO2Me

CO2Me H

CO2H

100% ee

Figure 6. Some in vitro enzyme reactions

Pure enzymes can be prohibitively expensive and difficult to obtain. Often it is more practical to use crude extracts or even the intact organisms which contain the desired enzyme. The best known example of this is probably the use of brewers yeast in the production of many forms of alcohol. But while commercially and socially this may be a significant process it is actually quite a trivial use of a powerful technique since alcohol contains no chiral centres and can be prepared more cheaply by conventional chemistry. A much more elegant example of using microorganisms is the commercial production of ephedrine and pseudoephedrine starting from benzaldehyde shown in Figure 7. Note that only the first step in the sequence, the introduction of the chiral centre, takes advantage
O H yeast OH O CH3NH2 OH N

of enzymes. Once the chiral centre is introduced, conventional chemical techniques become more cost effective. Ephedrine occurs naturally in some plants, extracts of which have been used by the Chinese for at least 2,000 years. It acts as a stimulant and bronchodilator. Pseudoephedrine is widely used in cold medicines as a nasal decongestant under a number of trade names including Sudafed and Sinufed. Both ephedrine and pseudoephedrine target the adrenergic receptors (the same receptors targeted by adrenaline) In general, a single enzyme only carries out one transformation. Only rarely do they catalyse two or more transformations and when they do it is usually caused by a conglomerate or complex of several different enzymes. Clearly then, the biosynthesis of most compounds will require a series of different enzymes and their corresponding cofactors. These series are usually referred to as biosynthetic, or biochemical pathways. The biosynthesis of adrenaline from the amino acid tyrosine (Figure 8) is a typical example of a biosynthetic pathway, involving several discrete steps, each requiring a different enzyme related cofactors. Adrenaline is responsible for the so called fight or flight response caused by a bad fright or dangerous situation. The biosynthesis of adrenaline takes place in the split second between getting a fright and the resulting rise in heart rate. An intermediate in this pathway, dopamine, is also an important neurotransmitter in the brain. Low levels of dopamine are associated with Parkinsons disease but treating sufferers of this disease with dopamine has no effect because it cannot cross the blood brain barrier, a protective membrane surrounding the brain. However, L-dopa has been used as a treatment because it can cross this barrier and once inside the brain it is decarboxylated to dopamine by the enzyme dopa decarboxylase. The enantiomer, D-dopa, cannot be used because of adverse side reactions.

OH H2 / Pd

OH H N Ac2O H+

H N

l-ephedrine

d-pseudoephedrine

Figure 7. Commercial production of ephedrine and pseudoephedrine using yeast

NH3 HO CO2
-

Appendix 2 - Enantiomeric Excess (ee)

L-tyrosine

Enantiomeric excess (ee) is a measure of the optical purity of a mixture of enantiomers. ee = (D-L) / (D+L) where D and L are the relative amounts of enantiomers.

tyrosine hydroxylase

HO HO

NH3 CO2
-

dopa

Appendix 3 - Common & Trade Names

dopa decarboxylase tetrahydropteridines Fe2+ O2 NH2 dopamine HO dopamine -oxidase ascorbate (vitamin C) Cu2+ O2 OH HO HO phenylethanolamine N-methyltransferase NH2 norepinephrine (noradrenaline)

HO

S-adenosylmethionine

OH HO HO NH Me epinephrine (adrenaline)

There are three different types of names applied to chemicals in the marketplace, be they pharmaceuticals, pesticides or whatever. There is the correct chemical name, but obviously this can at times be rather cumbersome and inappropriate. Therefore the use of a common name is essential. These common names should not be confused with Trade names. Trade names are made up by the company that markets the product. A single chemical can have dozens of trade names depending on the company, the country, the type of formulation, etc. However, to avoid any possible confusion it should have only one common name throughout the world. This common name must be approved by regulatory authorities and great care is taken to ensure the proposed name is suitable for all languages and countries. For example it must not be an existing product or word in any country, should be pronounceable in all languages and fulfil a number of other criteria.

Figure 8. The biosynthesis of adrenaline

transition states

E
uncatalysed

Appendix 1 - Catalysts
Catalysts, and this includes enzymes, act by lowering the activation energy (E) of the reaction. They do not affect E and therefore do not make energetically unfavourable reactions proceed, nor do they affect equilibrium concentrations. Note that when talking about enzymes the starting material is normally referred to as the substrate.
catalysed

substrate

E
product

reaction path