Biochemical Engineering

Lect. 2
 Enzymes
• Enzymes are biological catalysts that are
protein molecules in nature.
• They are produced by living cells (animal,
plant, and microorganism). and are absolutely
essential as catalysts in biochemical reactions.
• Almost every reaction in a cell requires the
presence of a specific enzyme.
• Function of enzymes in a living system is to
catalyze the making and breaking of chemical
bonds. they increase the rate of reaction
• The catalytic ability of enzymes is due to its
particular protein structure.
• A specific chemical reaction is catalyzed at a
small portion of the surface of an enzyme, which
is known as the active site.
• Some physical and chemical interactions occur at
this site to catalyze a certain chemical reaction
for a certain enzyme.
 Protein
• Proteins are polymers of amino acids
• Amino Acids
• Amino acids are the building blocks of proteins.
All AA’s have the same basic structure:
• For amino acids, the R-group is often called the
“side-chain” or “variant group.”
• The side-chain can be a hydrogen atom,
hydrocarbon, or various other groups of bonded
atoms.
• For example, if the side-chain is a hydrogen atom
(H), then the amino acid is called glycine; if the
side-chain is a methyl group (CH3), then the
amino acid is called alanine.
• Twenty different amino acids are found in
protein.
• They are called α-amino acids because their side-
chains are attached to α-carbons.
• different amino acids are classified based on the
side chain or R group
• The twenty common amino acids are often
referred to using three-letter abbreviations. The
structures, names, and abbreviations for the
twenty common amino acids are shown below.
Note that they are all α-amino acids.
 Classification
• Three systems of classifying amino acids are in vogue.
A. On the basis of the composition of the side chain or
R group.
• There are 20 different amino acids which regularly
appear in proteins.
• These possess a side chain which is the only variable
feature present in their molecules.
• The other features such as α-carbon, carboxyl group and
amino group are common to all the amino acids.
• The common component of an amino acid appears is
• Based on the composition of the side chain, the twenty amino
acids,, may be grouped into following 8 categories (Fairley and
Kilgour, 1966) :
I. Simple amino acids.
These have no functional group in the side chain, e.g., glycine,
alanine, valine, leucine and isoleucine.
II Hydroxy amino acids.
These contain a hydroxyl group in their side chain, e.g., serine and
threonine.
III Sulfur-containing amino acids.
these possess a sulfur atom in the side chain, e.g., cysteine and
methionine.
IV Acidic amino acids.
These have a carboxyl group in the side chain, e.g., aspartic acid and
glutamic acid.
V. Amino acid amides.
These are derivatives of acidic amino acids in which one of the
carboxyl group has been transformed into an amide group (-
CO.NH2), e.g., asparagine and glutamine.
VI Basic amino acids.
These possess an amino group in the side chain, e.g., lysine and
arginine.
VII Heterocyclic amino acids.
These amino acids have in their side chain a ring which possesses at
least one atom other than the carbon, e.g., tryptophan, histidine and
proline.
VIII Aromatic amino acids.
These have a benzene ring in the side chain, e.g., phenylalanine and
tyrosine.

• The classification given above is only a practical one and can conveniently be
followed. It does not, however, strictly delimit the various categories. For
example, tryptophan may also be included under aromatic amino acids and
similarly, histidine under basic amino acids.
B. On the basis of the number of amino and
carboxylic groups. (McGilvery and Goldstein(1979))
have classified various amino acids as follows :
I. Monoamino-monocarboxylic amino acids :
1. Unsubstituted Glycine Alanine, Valine, Leucine, Isoleucine
2. Heterocyclic Proline
3. Aromatic Phenylalanine, Tyrosine,Tryptophan
4. Thioether Methionine.
5. Hydroxy Serine, Threonine
6. Mercapto Cysteine
7. Carboxamide Asparagine, Glutamine
II. Monoamino-dicarboxylic amnino acids : Aspartic acid,
Glutamic acid
III. Diamino-monocaryboxylic amino acids : Lysine, Arginine,
Histidine
C. On the basis of polarity of the side chain or
R group.
• A more meaningful classification of amino acids is,
however, based on the polarity of the R groups
present in their molecules, i.e., their tendency to
interact with water at biological pH (near pH 7.0).
• The R groups of the amino acids vary widely with
respect to their polarity from totally nonpolar or
hydrophobic (water-hating) R groups to highly polar
or hydrophilic (water-loving) R groups.
• This classification of amino acids emphasizes the
possible functional roles which they perform in
proteins.
• The system recognizes following 4 categories :
1. Amino acids with nonpolar R groups.
• The R groups in this category of amino acids are
hydrocarbon in nature and thus hydrophobic.
• This group includes five amino acids with aliphatic R groups
(alanine, valine, leucine isoleucine, proline), two with aromatic
rings (phenylalanine, tryptophan) and one containing sulfur
(methionine).
II. Amino acids with polar but uncharged R groups.
• The R groups of these amino acids are more soluble in water
i.e., more hydrophilic than those of the nonpolar amino
acids because they contain functional groups that form
hydrogen bonds with water.
• This category includes 7 amino acids, viz., glycine, serine,
threonine, tyrosine, cysteine, asparagine and glutamine.
• The polarity of these amino acids may be due to either a
hydroxyl group (serine, threonine, tyrosine) or a sulfhydryl
group (cysteine) or an amide group (asparagine, glutamine).
III. Amino acids with negatively charged (= acidic) R
groups.
• These are monoamino-dicarboxylicacids. In other
words, their side chain contains an extra carboxyl
group with a dissociable
• proton. The resulting additional negative charge
accounts for the electrochemical behavior of proteins.
• The two amino acids which belong to this category
are aspartic and glutamic.
IV. Amino acids with positively charged (=basic) R
groups.
• These are Diamino-Monocarboxylic acids. In other
words, their side chain contains an extra amino
group which imparts basic properties to them. As
Lysine, arginine and histidine.
NONSTANDARD PROTEIN AMINO ACIDS
• As an example, hydroxyproline has a limited
distribution in nature but constitutes as much as
12% of the composition of collagen, an important
structural protein of animals.
• Similarly, hydroxylysine is also a component of
collagen, where it accounts for about 1% of the total
amino acids. N-methyllysine is found in myosin, a
contractile protein of muscle.
• Another important nonstandard or less common
amino acid is γ-carboxyglutamate, which is found in
the blood-clotting protein, prothrombin as well as in
certain other proteins that bind Ca2+ in their
biological function.
 NONPROTEIN AMINO ACIDS
• There are some 300 additional amino acids
which are never found as constituents of
proteins but which either play metabolic roles
or occur as natural products.
• Among the important nonprotein amino acids,
which play metabolic roles, are L-ornithine, L-
citrulline, β-alanine, creatine and γ-
aminobutyrate. L-ornithine and L-citrulline
occur in free state in the animal tissues and
are metabolic intermediates in the urea cycle.
 The Peptide Bond
• To make a protein, these amino acids are
joined together in a polypeptide chain
through the formation of a peptide bond.
 Polypeptides
• Proteins are nothing more than long polypeptide
chains.
• Chains that are less than 40-50 amino acids or
residues
• are often referred to as polypeptide chains since
they are too smal to form a functional domain.
• Larger than this size, they are called proteins
• The structure, function and general properties of
a protein are all determined by the sequence of
amino acids that make up its primary sequence.
CHEMICAL BONDS INVOLVED IN
PROTEIN STRUCTURE

Types of chemical bonds involved in protein structure

A. Primary Bond
• The principal linkage found in all proteins is the
covalent peptide bond, -CO-NH-
• It is a specialized amide linkage where C atom of
-COOH group of one amino acid is linked with
the N atom of -NH2 group of the adjacent amino
acid. Peptide bond is, in fact, the backbone of
the protein chain.
B. Secondary Bonds
• hold the chain in its natural configuration. Some
of the secondary bonds commonly found in
proteins are listed below :
1. Disulfide Bond (-S-S-).
Covalent bond found between amino acid
residues in proteins and polypeptides is the
disulfide bond, which is formed by the oxidation
of the thiol or sulfhydryl (-SH) groups of two
cysteine residues to yield a mole of cystine, an
amino
2. Hydrogen Bond (>CO......HN<).
• When a group containing a hydrogen atom, that is
covalently-bonded to an electronegative atom, such
as oxygen or nitrogen,
• The formation of a hydrogen bond is due to the
tendency of hydrogen atom to share electrons with
two neighbouring atoms, esp., O and N. For example,
• The carbonyl oxygen of one peptide bond shares its
electrons with the hydrogen atom of another peptide
bond. Thus, An interaction sets in between a C=O
group and the proton of an NH or OH group if these
groups come within a distance of about 2.8 Å. This
secondary valence bond is symbolized by a dotted
line,
3. Nonpolar or Hydrophobic Bond.
Many amino acids (like alanine, valine, leucine,
isoleucine, methionine, tryptophan, phenylalanine
and tyrosine) have the side chains or R groups
which are essentially hydrophobic
Such R groups can unite among themselves with
elimination of water to form linkages between
various segments of a chain or between different
chains.
This is very much like the coalescence of oil
droplets suspended in water.
4. Ionic or Electrostatic Bond or Salt linkage or
Salt bridge.
Ions possessing similar charge repel each other
whereas the ions having different charges
attract each other.
For example, Divalent cations like magnesium
may form electrostatic bonds with 2 acidic side
chains, interaction between the acidic and basic
groups
 Enzyme structure
• Enzymes have four levels of structures These are:
A. Primary structure
B. Secondary structure
C. Tertiary structure
D. Quaternary structure
• The enzyme structure ranges from a basic amino acid
sequence to a three dimensional (3D) structure in a folded
protein.
• The amino acid sequence in polypeptide chains in each
enzyme is distinct and determines the three-dimensional
shape.
• 3D structure of an enzyme that determines the enzyme
activities.
• We will look at these structures in detail in the sections
below.
1. Primary Structure :
The sequence of amino acids in an enzyme is the primary
structure.

In the primary structure, the constituent amino acids are

linked by peptide bonds (-CONH-) bonds
 Secondary structure
• The secondary structure in enzymes refers to the
interaction of amino acids in a chain (primary structure)
which are closely located.
• There are two types of secondary structures: helical
(called α helices) and pleated sheets (called β pleated
sheets).
• Alpha helix
• The alpha helix is a helical structure, coiled around an
axis.
• The helix is right-handed in nature.
• The alpha helix is characterized by intermolecular
hydrogen bonds between the O atom of the C=O of each
peptide bond in the strand and the N-H group of the
peptide bond
• The side-chain substituents of the amino acids
extend to the outside from the helix.
• The helix has about 3.6 amino acids per turn
on an average, meaning that it will have 36
amino acids in 10 turns. The pitch is 5.4 Å
• Alpha helices form more readily in enzymes
than any other possible conformations owing
to the optimal use of internal hydrogen bonds
is made in these arrangements for attaining
stability.
 Beta pleated sheet
• The second form of secondary structure in
enzymes is the beta pleated sheet.
• This structure is formed by intermolecular
hydrogen bonding between two or more straight
chains.
• The O atom of the C=O of peptide bond in one
strand hydrogen bonds with the N-H group of
the peptide bond in an adjacent strand.
• Again, the two strands involved in the formation
of beta pleated sheets can run either parallel to
each other or anti-parallel to each other.
• If the amino groups of both chains are on the
same side, the sheet are said to be parallel to
each other.
• On the other hand, if the amino groups of
both chains are on the opposite side, the
chains are said to run in the opposite
direction. In this case, the sheet is termed
antiparallel.
• The anti-parallel ß-sheet is more stable than
parallel sheet owing greater alignment in the
hydrogen bonds.
Beta Sheets
 Tertiary structure
• The protein molecule arranges itself three
dimensionally in such a way as to achieve low
energy and maximum stability.
• The various interactions involved in the
formation/stabilization of a tertiary structure
are Hydrogen bonds, polar-polar interaction,
hydrophobic interaction, ionic interaction,
formation of disulfide bonds, Van der Waals
forces.
• the side chains of amino acids which are
hydrophobic in nature such as phenylalanine or
isoleucine, tend to remain buried within the
protein/enzyme core, owing to their minimal
affinity for the aqueous medium.
• The alkyl groups of Ala, Val, Leu, Ileu often form
hydrophobic interactions between one-another.
• Acidic or basic amino acid side-chains are polar
in nature, and therefore remain exposed on the
enzyme surface, to allow for greater water
solubility.
 Quaternary structure
• proteins or functional enzymes can be made up
of more than one polypeptide chains, which are
known as subunits.
• The interaction between these subunits is called
the quaternary structure.
• Various interactions, including H-bonding,
disulfide-bridges and salt bridges are also
involved in stabilizing the overall complex.
• Folded proteins then bind together to form
• dimer, trimers, or higher order structures
• The functional form of hemoglobin is a tetramer
 Nomenclature of enzyme
• Originally enzymes were given non descriptive
names such as:
• rennin curding of milk to start cheese-making
processor
• pepsin hydrolyzes proteins at acidic pH
• trypsin hydrolyzes proteins at mild alkaline pH
• The nomenclature was later improved by adding
the suffix -ase to the name of the substrate with
which the enzyme functions, or to the reaction
that is catalyzed.
• For example:
 Commercial application of enzeme
• The enzymes produced commercially can be classified
into three major categories (Crueger and Crueger, 1984):
1. Industrial enzymes,
such as amylases, proteases, glucose isomerase, lipase,
catalases, and penicillin acylases
2. Analytical enzymes,
such as glucose oxidase, galactose oxidase, alcohol
dehydrogenase, hexokinase, muramidase, and cholesterol
oxidase
3. Medical enzymes,
such as asparaginase, proteases, lipases, and streptokinase
a-amylase, glucosamylase, and glucose isomerase serve
mainly to convert starch into high-fructose corn syrup
(HFCS), as follows:
Thank you