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PART
A
DRUG TARGETS
2. Protein and Enzymes: structure and function

TOPIC
2 Protein structure and function
2.1 The primary structure of proteins; 2.2 The secondary structure of proteins
2.3 The tertiary structure of proteins; 2.4 The quaternary structure of proteins
2.5 Translation and post-translational modifications 25
2.6 Proteomics ; 2.7 Protein function
3 Enzymes: structure and function
3.1 Enzymes as catalysts; 3.2 How do enzymes catalyse reactions?;
3.3 The active site of an enzyme; 3.4 Substrate binding at an active site;
3.5 The catalytic role of enzymes;3.6 Regulation of enzymes;
3.7Isozymes; 3.8 Enzyme kinetics
Drug targets

o Medicinal chemistry is the study of how novel drugs can be designed and
developed.
o The major drug targets are normally large molecules (macromolecules), such as
proteins and nucleic acids. Knowing the structures, properties, and functions of
these macromolecules is crucial if we are to design new drugs.
o There are a variety of reasons for this:
• Firstly, it is important to know what functions different macromolecules have in
the body and whether targeting them is likely to have a beneficial effect in
treating a particular disease.
• Secondly, a knowledge of macromolecular structure is crucial if one is to design
a drug that will bind effectively to the target.
• Thirdly, a drug must not only bind to the target, it must bind to the correct
region of the target.
• Finally, an understanding of how a macromolecule operates is crucial if one is
going to design an effective drug that will interfere with that process.
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2.1 The primary structure of proteins

The primary structure is the order in which the

individual amino acids making up the protein are
linked together through peptide bonds
The primary structure of Met-enkephalin (one of the
body’s own painkillers) is shown in Fig. 2.2.

FIGURE 2.2 Met-enkephalin. The short hand notation for this

peptide is H-Tyr-Gly-Gly-Phe-Met-OH or YGGFM.

The peptide bond in proteins is planar in nature as a

result of the resonance structure shown in Fig. 2.3.
This gives the peptide bond a significant double bond
character, which prevents rotation. As a result, bond
rotation in the protein backbone is only possible for
the bonds on
2.2 The secondary structure of proteins

FIGURE 2.5 The α-helix for proteins showing intramolecular FIGURE 2.6 The β-pleated sheet (antiparallel
hydrogen bonds and the position of side chains. arrangement).
2.3 The tertiary structure of proteins

FIGURE 2.7 Hydrogen bonding in antiparallel and parallel

β-sheets (the arrows are pointing to the C-terminal

FIGURE 2.9 The pdb file (1hcl) for human cyclin-

dependent kinase 2 (CDK2), where cylinders
represent α-helices and arrows represent β-sheets.
FIGURE 2.8 The β-turn showing A pdb file contains the three-dimensional structural
hydrogen bonding between the first information for a protein and can be downloaded
and third peptide bond. from the Brookhaven protein data bank. Each
protein structure file is given a code, for example
1hcl.
FIGURE 2.10 Tertiary structure formation as
a result of intramolecular interactions.
2.3.1 Covalent bonds—disulphide links

2.3.2 Ionic or electrostatic bonds

2.3.3 Hydrogen bonds
Hydrogen bonds can be viewed as a weak form
of ionic interaction as they involve interactions
between atoms having partial charges.

They can be formed between a large number of

amino acid side chains, such as serine,
threonine, aspartic acid, glutamic acid,
glutamine, lysine, arginine, histidine, tryptophan,
tyrosine, and asparagine.

Two examples are shown in Fig. 2.13.

2.3.4 Van der Waals and hydrophobic interactions
Van der Waals interactions are weaker interactions than
hydrogen bonds and can take place between two hydro-
phobic regions of the protein. For example, they can
take place between two alkyl groups (Fig. 2.14).

The amino acids alanine, valine, leucine, isoleucine,

phenylalanine, and proline all have hydrophobic side
chains capable of interacting with each other by van der
Waals interactions.

The side chains of other amino acids, such as

methionine, tryptophan, threonine, and tyrosine,
contain polar functional groups, but the side chains also
have a substantial hydrophobic character and so van der
Waals interactions are also possible for these amino
acids. Hydrophobic interactions (section 1.3.6) are also
important in the coming together of hydrophobic
residues.
2.3.5 Relative importance of bonding interactions
We might expect the relative importance of the bonding
interactions in protein tertiary structure to follow the same
order as their strengths: covalent, ionic, hydrogen bonding,
and, finally, van der Waals. In fact, the opposite is generally
true. Usually, the most important bonding interactions are
those due to van der Waals interactions and hydrogen
bonding, while the least important interactions are those due
to covalent and ionic bonding.

2.3.6 Role of the planar peptide bond

Planar peptide bonds indirectly play an important role in tertiary structure. Bond rotation in
peptide bonds is hindered, with the trans conformation generally favoured, so the number
of possible conformations that a protein can adopt is significantly restricted, making it more
likely that a specific conformation is adopted. Polymers without peptide bonds do not fold
into a specific conformation, because the entropy change required to form a highly ordered
structure is extremely unfavourable. Peptide bonds can also form hydrogen bonds with
amino acid side chains and play a further role in determining tertiary structure.
2.4 The quaternary structure of proteins

Only proteins that are made up of a

number of protein subunits have
quaternary structure.

For example, haemoglobin is made up of

four protein molecules—two identical
alpha subunits and two identical beta
subunits (not to be confused with the
alpha and beta terminology used in
secondary structure). The quaternary
structure of haemoglobin is the way in
which these four protein units associate
with each other.
As this must inevitably involve interactions between the exterior surfaces of proteins, ionic
bonding can be more important to quaternary structure than it is to ter- tiary structure.
Nevertheless, hydrophobic and van der
Waals interactions have a role to play. It is not possible for a protein to fold up such that all its
hydrophobic groups are placed towards the centre. Some of these groups may be stranded on
the surface.

If they form a small hydropho- bic area on the protein surface, there is a distinct advan- tage
for two protein molecules to form a dimer such that the two hydrophobic areas face each
other rather than be exposed to an aqueous environment (Fig. 2.17).
2.5 Translation and post- translational modifications
The process by which a protein is synthesized in the cell is called translation. Many proteins
are modified following translation (Fig. 2.18), and these modifications can have wide-ranging
effects.

For example, the N-terminals of many

proteins are acetylated, making these
proteins more resistant to degradation.
Acetylation of proteins also has a role to
play in the control of transcription, cell
proliferation, and differentiation (section
21.7.3).

The fibres of collagen are stabilized by the hydroxyla- tion of proline

residues. Insufficient hydroxylation results in scurvy (caused by a deficiency
of vitamin C).
The glutamate residues of prothrombin, a clotting protein, are carboxylated to form γ-
carboxyglutamate structures. In cases of vitamin K deficiency, carboxylation does not occur
and excessive bleeding results.

The serine, threonine, and tyrosine residues of many proteins

are phosphorylated and this plays an important role in
signalling pathways within the cell (sections 5.2–5.4).
 Several proteins are cleaved into smaller proteins
or peptides following translation. For example, the
enkephalins are small peptides which are derived
from proteins in this manner (section 24.8). Active
enzymes are sometimes formed by cleaving a
larger protein precursor. Often, this serves to
protect the cell from the indiscriminate action of
an enzyme. For example, digestive enzymes are
stored in the pancreas as inactive protein
FIGURE 2.18 Examples of post-translational precursors and are only produced once the protein
modifications carried out on proteins. precursor is released into the intestine. In blood
clotting, the soluble protein fibrinogen is cleaved
to insoluble fibrin when the latter is required.
Many of the proteins present on the surface of cells are Some polypeptide hormones are also produced
linked to carbohydrates through asparagine residues. from the cleavage of protein precur- sors. Finally,
Such carbohydrates are added as post-translational mod- the cleavage of a viral polyprotein into con-
ifications and are important to cell–cell recognition, dis- stituent proteins is an important step in the life
ease processes, and drug treatments (section 10.7). The cycle of the HIV virus and has proved a useful
target for several drugs currently used to combat
proteins concerned are called glycoproteins or proteo-
AIDS (section 20.7.4).
glycans, and are members of a larger group of molecules
called glycoconjugates.
2.6 Proteomics
A lot of publicity has been rightly accorded to the Human Genome Project, which has now
been completed. The science behind this work is called genomics and it involves the
identification of the genetic code in humans and other species. The success of this work has
been hailed as a breakthrough that will lead to a new era in medicinal research.

Proteomics is far more challenging than genomics because of the complexity of interactions
that can take place between proteins (see Chapter 5). Moreover, the pattern and function of
proteins present in a cell depend on the type of cell it is and this pattern can alter in the
diseased state. Nevertheless, the race is now on to analyse the structure and function of
proteins, many of which are completely new to science, and to see whether they can act as
novel drug targets for the future.

Identifying the proteins present in a cell usually involves analysing the contents of the cell
and separating out the proteins using a technique known as 2Dl gel electrophoresis. Mass
spectrometry can then be used to study the molecular weight of each protein. Assuming a
pure sample of protein is obtained, its pri-mary structure can be identified by traditional
sequencing techniques. The analysis of secondary and tertiary structures is trickier.
2.7 Protein function
We are now ready to discuss the various types of protein which act as drug targets.

2.7.1 Structural proteins

Structural proteins do not normally act as drug targets. However, the structural protein tubulin is
an exception. Tubulin molecules polymerize to form small tubes called microtubules in the cell’s
cytoplasm (Fig 2.19). These microtubules have various roles within the cell, including the
maintenance of shape, exocytosis, and release of neurotransmitters. They are also involved in
the mobility of cells. For example, inflammatory cells called neutrophils are mobile cells which
normally protect the body against infection. However, they can also enter joints, leading to
inflammation and arthritis.
Tubulin is also crucial to cell division. When a cell is about to divide, its microtubules
depolymerize to give tubulin. The tubulin is then re-polymerized to form a structure called
a spindle which then serves to push apart the two new cells and to act as a framework on
which the chromosomes of the original cell are transferred to the nuclei of the daughter
cells (Fig. 2.20). Drugs that target tubulin and inhibit this process are useful anticancer
agents (section 10.2.2).

The structural proteins of viruses are important to the survival of the virus outside their host cell.
Some of these proteins are proving to be interesting drug targets for the design of new antiviral
agents and are discussed in more detail in sections 20.7.5 and 20.9.
2.7.2 Transport proteins
Transport proteins are present in the cell membrane and act as the cell’s ‘smugglers’—
smuggling the important chemical building blocks of amino acids, sugars, and nucleic acid
bases across the cell membrane such that the cell can synthesize its proteins,
carbohydrates, and nucleic acids. They are also important in transporting important
neurotransmitters (section 4.2) back into the neuron that released them so that the
neurotransmitters only have a limited period of activity.
2.7.3 Enzymes and receptors

The most important drug targets in medicinal chem- istry are enzymes and receptors.
Chapters 3 and 4 are devoted to the structure and function of these proteins respectively.

2.7.4 Miscellaneous proteins and protein–protein interactions

There are many situations in cell biology where proteins are required to interact with each
other in order to produce a particular cellular effect. We have already seen an example of
this in the polymerization of tubulin proteins in order to form microtubules (section 2.7.1).

The structures of many important drug targets, such as ion channels, enzymes, and receptors
consist of two or more protein subunits associated with each other. The signal transduction
processes described in Chapter 5 show many instances where a variety of proteins, such as
receptors, signal proteins, and enzymes, associate with each other in order to transmit a
chemical signal into the cell. The actions of insulin are mediated through a pro- tein–protein
interaction (section 4.8.3).
QUESTIONS

1. Draw the full structure of L-alanyl-L-phenylalanyl-glycine.

2. What is unique about glycine compared with other naturally-occurring amino acids?
3. Identify the intermolecular/intramolecular interactions
that are possible for the side chains of the following amino acids; serine,
phenylalanine, glycine, lysine, aspartic acid, and aspartate.
4. The chains of several cell membrane-bound proteins wind back and forth through the
cell membrane, such that some parts of the protein structure are extracellular, some
parts are intracellular, and some parts lie within the cell membrane. How might the
primary structure of a protein help in distinguishing the portions of the protein
embedded within the cell membrane from those that are not?
5. What problems might you foresee if you tried to synthesize L-alanyl-L-valine directly
from its two component amino acids?
6. The tertiary structure of many enzymes is significantly altered by the
phosphorylation of serine, threonine, or tyrosine residues. Identify the functional
groups that are involved in these phosphorylations and suggest why
phosphorylation affects tertiary structure.
7. What is the one-letter code for the polypeptide Glu–Leu– Pro–Asp–Val–Val–Ala–
Phe–Lys–Ser–Gly–Gly–Thr?