BLBS102-c07 BLBS102-Simpson March 21, 2012 11:12 Trim: 276mm X 219mm Printer Name: Yet to Come
7 Biocatalysis, Enzyme Engineering and Biotechnology 127feature of all 20 amino acids is a central carbon atom (Cα)to
which a hydrogen atom, an amino group (–NH 2 ) and a carboxy
group (–COOH) are attached. Free amino acids are usually zwit-
terionic at neutral pH, with the carboxyl group deprotonated and
the amino group protonated. The structure of the most common
amino acids found in proteins is shown in Table 7.1. Amino
acids can be divided into four different classes depending on the
structure of their side chains, which are called R groups: non-
polar, polar uncharged, negatively charged (at neutral pH) and
positively charged (at neutral pH; Richardson 1981). The prop-
erties of the amino acid side chains determine the properties of
the proteins they constitute. The formation of a succession of
peptide bonds generates the ‘main chain’ or ‘backbone’.
The primary structure of a protein places several constrains
on how it can fold to produce its three-dimensional structure
(Cantor 1980, Fersht 1999). The backbone of a protein consists
of a carbon atom Cαto which the side chain is attached, a NH
group bound to Cαand a carbonyl group C=O, where the
carbon atom C is attached to Cα(Fig. 7.1) The peptide bond is
planar since it has partial (∼40%) double bond character with
πelectrons shared between the C–O and C–N bonds (Fig. 7.1).
The peptide bond has atrans-conformation, that is the oxygen of
the carbonyl group and the hydrogen of the NH group are in the
transposition; thecisconformation occurs only in exceptional
cases (Richardson 1981).
Enzymes have several ‘levels’ of structure. The protein’s se-
quence, that is the order of amino acids, is termed as its ‘primary
structure’. This is determined by the sequence of nucleotide
bases in the gene that codes for the protein. Translation of the
mRNA transcript produces a linear chain of amino acids linked
together by a peptide bond between the carboxyl carbon of the(A)(B)Figure 7.1.(A) The amide bond showing delocalisation of
electrons. (B) A tripeptide unit of a polypeptide chain showing the
planar amide units and the relevant angles of rotation about the
bonds to the centralα-carbon atom.first amino acid and the free amino group of the second amino
acid. The first amino acid in any polypeptide sequence has a free
amino group and the terminal amino acid has a free carboxyl
group. The primary structure is responsible for the higher levels
of enzyme’s structure and therefore for the enzymatic activity
(Richardson 1981, Price and Stevens 1999).The Three-Dimensional Structure of EnzymesEnzymes are generally very closely packed globular structures
with only a small number of internal cavities, which are normally
filled by water molecules. The polypeptide chains of enzymes
are organised into ordered, hydrogen bonded regions, known
assecondary structure(Andersen and Rost 2003). In these or-
dered structures, any buried carbonyl oxygen forms a hydro-
gen bond with an amino NH group. This is done by forming
α-helices andβ-pleated sheets, as shown in Figure 7.2. Theα-
helix can be thought of as having a structure similar to a coil or
spring (Surewicz and Mantsch 1988). Theβ-sheet can be visu-
alised as a series of parallel strings laid on top of an accordion-
folded piece of paper. These structures are determined by the
protein’s primary structure. Relatively small, uncharged, polar
amino acids readily organise themselves intoα-helices, while
relatively small, non-polar amino acids formβ-sheets. Proline is
a special amino acid because of its unique structure (Table 7.1).
Introduction of proline into the sequence creates a permanent
bend at that position (Garnier et al. 1990). Therefore, the pres-
ence of proline in anα-helix orβ-sheet disrupts the secondary
structure at that point. The presence of a glycine residue confers
greater than normal flexibility on a polypeptide chain. This is
due to the absence of a bulky side chain, which reduces steric
hindrance.
Another frequently observed structural unit is theβ-turn (Fang
and Shortle 2003). This occurs when the main chain sharply
changes direction using a bend composed of four successive
residues, often including proline and glycine. In these units,
the C=O group of residue i is hydrogen bonded to the NH of
residue i+3 instead of i+4asinα-helix. Many different types of
β-turn have been identified, which differ in terms of the number
of amino acids and in conformation (e.g. Type I, Type II, Type
III; Sibanda et al. 1989).
The three-dimensional structure of a protein composed of
a single polypeptide chain is known as itstertiary structure.
Tertiary structure is determined largely by the interaction of
R groups on the surface of the protein with water and with
other R groups on the protein’s surface. The intermolecular non-
covalent attractive forces that are involved in stabilising the
enzyme’s structure are usually classified into three types: ionic
bonds, hydrogen bonds and van der Waals attractions (Matthews
1993). Hydrogen bonding results from the formation of hydro-
gen bridges between appropriate atoms; electrostatic bonds are
due to the attraction of oppositely charged groups located on
two amino acid side chains. Van der Waals bonds are gener-
ated by the interaction between electron clouds. Another im-
portant weak force is created by the three-dimensional structure
of water, which tends to force hydrophobic groups together in
order to minimise their disruptive effect on hydrogen-bonded