Download Polar amino acids with negative charge

Dr. Gobinath Pandian
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Chapter: 1
Introduction Amino acids
Introduction to Proteins &
Enzymes
INTRODUCTION
Amino acids are molecules containing an amine
group (NH), a carboxylic acid group (COOH) and a
side chain(R) that varies between different amino
acids. These molecules contain the key elements
of carbon, hydrogen, oxygen, and nitrogen.
• Any of a class of organic compounds in which a carbon atom
has bonds to an amino group (-NH2), a carboxyl group (-COOH),
a hydrogen atom (-H), and an organic side group (called -R).
• Amino acids are organic compounds made of carbon,
hydrogen, oxygen, nitrogen, and (in some cases) sulfur bonded
in characteristic formations.
• They are therefore both carboxylic acids and amines. The
physical and chemical properties unique to each result from
the properties of the R group, particularly its tendency to
interact with water and its charge (if any).
• Amino acids joined linearly by peptide bonds (see covalent
bond) in a particular order make up peptides and proteins.
• Amino acids are critical to life, and have
many functions in metabolism. One
particularly important function is to serve as
the building blocks of proteins, which are just
linear chains of amino acids, or more
precisely, amino acid residues.
• Every protein is chemically defined by the
order of amino acid residues, their primary
structure and this, in turn, determines their
secondary structure
Glycine is the smallest of the amino acids. It is ambivalent, meaning that it
can be inside or outside of the protein molecule. In aqueous solution at or
near neutral pH, glycine will exist predominantly as the zwitterion
The isoelectric point or isoelectric pH of glycine will be centered between the
pKas of the two ionizable groups, the amino group and the carboxylic acid
group.
In estimating the pKa of a functional group, it is important to consider the
molecule as a whole. For example, glycine is a derivative of acetic acid, and
the pKa of acetic acid is well known. Alternatively, glycine could be considered
a derivative of aminoethane.
Cysteine is one of two sulfur-containing amino acids; the other is methionine.
Cysteine differs from serine in a single atom-- the sulfur of the thiol replaces
the oxygen of the alcohol.
The amino acids are, however, much more different in their physical and
chemical properties than their similarity might suggest.
Cysteine also plays a key role in stabilizing extracellular proteins. Cysteine can
react with itself to form an oxidized dimer by formation of a disulfide bond.
The environment within a cell is too strongly reducing for disulfides to form,
but in the extracellular environment, disulfides can form and play a key role in
stabilizing many such proteins, such as the digestive enzymes of the small
intestine.
• Methionine, an essential amino acid, is one of the two sulfurcontaining amino acids. The side chain is quite hydrophobic and
methionine is. usually found buried within proteins.
• Unlike cysteine, the sulfur of methionine is not highly
nucleophilic, although it will react with some electrophilic centers.
It is generally not a participant in the covalent chemistry that
occurs in the active centers of enzymes.
• Methionine as the free amino acid plays several important roles in
metabolism. It can react to form S-Adenosyl-L-Methionine (SAM)
which servers at a methyl donor in reactions
• Alanine is a hydrophobic molecule. It is ambivalent,
meaning that it can be inside or outside of the protein
molecule. The α carbon of alanine is optically active; in
proteins, only the L-isomer is found.
• Note that alanine is the α-amino acid analog of the αketo acid pyruvate, an intermediate in sugar metabolism.
Alanine and pyruvate are interchangeable by a
transamination reaction.
• Asparagine is the amide of aspartic acid. The
amide group does not carry a formal charge under
any biologically relevant pH conditions. The amide
is rather easily hydrolyzed, converting asparagine
to aspartic acid.
• Asparagine has a high propensity to hydrogen
bond, since the amide group can accept two and
donate two hydrogen bonds.
• Asparagine is a common site for attachment of
carbohydrates in glycoproteins.
• Aspartic acid is one of two acidic amino acids. Aspartic acid and
glutamic acid play important roles as general acids in enzyme
active centers, as well as in maintaining the solubility and ionic
character of proteins.
• Proteins in the serum are critical to maintaining the pH balance
in the body; it is largely the charged amino acids that are
involved in the buffering properties of proteins.
• Aspartic acid and oxaloacetate are interconvertable by a simple
transamination reaction, just as alanine and pyruvate are
interconvertible.
• Glutamine is the amide of glutamic acid, and is uncharged
under all biological conditions.
• The additional single methylene group in the side chain
relative to asparagine allows glutamine in the free form or
as the N-terminus of proteins to spontaneously cyclize and
deamidate yielding the six-membered ring structure
pyrrolidone carboxylic acid, which is found at the Nterminus of many immunoglobulin polypeptides. This
causes obvious difficulties with amino acid sequence
determination.
• Histidine, an essential amino acid, has as a positively
charged imidazole functional group.
• The imidazole makes it a common participant in enzyme
catalyzed reactions.
• The unprotonated imidazole is nucleophilic and can serve
as a general base, while the protonated form can serve as
a general acid. The residue can also serve a role in
stabilizing the folded structures of proteins.
• Isoleucine, an essential amino acid, is one of the three amino acids
having branched hydrocarbon side chains. It is usually
interchangeable with leucine and occasionally with valine in
proteins.
• The side chains of these amino acids are not reactive and therefore
not involved in any covalent chemistry in enzyme active centers.
• However, these residues are critically important for ligand binding
to proteins, and play central roles in protein stability. Note also that
the β carbon of isoleucine is optically active, just as the β carbon of
threonine. These two amino acids, isoleucine and threonine, have in
common the fact that they have two chiral centers.
• Leucine, an essential amino acid, is one of the three
amino acid with a branched hydrocarbon side chain.
• It has one additional methylene group in its side chain
compared with valine.
• Like valine, leucine is hydrophobic and generally buried
in folded proteins.
• Lysine. an essential amino acid, has a positively charged ε-amino
group (a primary amine).
• Lysine is basically alanine with a propylamine substituent on
theβcarbon. The ε-amino group has a significantly higher pKa (about
10.5 in polypeptides) than does the α-amino group.
• The amino group is highly reactive and often participates in a
reactions at the active centers of enzymes. Proteins only have one α
amino group, but numerous ε amino groups
• Phenylalanine, an essential amino acid, is a derivative of
alanine with a phenyl substituent on the β carbon.
Phenylalanine is quite hydrophobic and even the free amino
acid is not very soluble in water.
• Due to its hydrophobicity, phenylalanine is nearly always found
buried within a protein. The π electrons of the phenyl ring can
stack with other aromatic systems and often do within folded
proteins, adding to the stability of the structure.
• Proline shares many properties with the aliphatic group.
• Proline is formally NOT an amino acid, but an imino acid.
Nonetheless, it is called an amino acid. The primary amine on
the α carbon of glutamate semialdehyde forms a Schiff base
with the aldehyde which is then reduced, yielding proline.
• When proline is in a peptide bond, it does not have a hydrogen
on the α amino group, so it cannot donate a hydrogen bond to
stabilize an α helix or a β sheet. It is often said, inaccurately,
that proline cannot exist in an α helix. When proline is found in
an α helix, the helix will have a slight bend due to the lack of
the hydrogen bond.
• Serine differs from alanine in that one of the
methylenic hydrogens is replaced by a hydroxyl
group.
• Serine is one of two hydroxyl amino acids. Both are
commonly considered to by hydrophilic due to the
hydrogen bonding capacity of the hydroxyl group.
• Threonine, an essential amino acid, is a hydrophilic molecule.
• Threonine is an other hydroxyl-containing amino acid. It differs
from serine by having a methyl substituent in place of one of
the hydrogens on the β carbon and it differs from valine by
replacement of a methyl substituent with a hydroxyl group.
• Note that both the α and β carbons of threonine are optically
active.
• Tryptophan, an essential amino acid, is the largest of
the amino acids. It is also a derivative of alanine,
having an indole substituent on the β carbon.
• The indole functional group absorbs strongly in the
near ultraviolet part of the spectrum. The indole
nitrogen can hydrogen bond donate, and as a result,
tryptophan, or at least the nitrogen, is often in contact
with solvent in folded proteins.
• Tyrosine, an essential amino acid, is also an aromatic amino acid and is
derived from phenylalanine by hydroxylation in the para position.
•
While tyrosine is hydrophobic, it is significantly more soluble that is
phenylalanine. The phenolic hydroxyl of tyrosine is significantly more
acidic than are the aliphatic hydroxyls of either serine or threonine,
having a pKa of about 9.8 in polypeptides.
•
As with all ionizable groups, the precise pKa will depend to a major
degree upon the environment within the protein.
• Tyrosines that are on the surface of a protein will generally have a lower
pKa than those that are buried within a protein; ionization yielding the
phenolate anion would be exceedingly unstable in the hydrophobic
interior of a protein.
• Valine, an essential amino acid, is hydrophobic, and as
expected, is usually found in the interior of proteins.
• Valine differs from threonine by replacement of the
hydroxyl group with a methyl substituent. Valine is often
referred to as one of the amino acids with hydrocarbon
side chains, or as a branched chain amino acid.
• Note that valine and threonine are of roughly the same
shape and volume. It is difficult even in a high resolution
structure of a protein to distinguish valine from
threonine.
• Glutamic acid has one additional methylene group in its side chain
than does aspartic acid. The side chain carboxyl of aspartic acid is
referred to as the β carboxyl group, while that of glutamic acid is
referred to as the γ carboxyl group.
• The pKa of the γ carboxyl group for glutamic acid in a polypeptide is
about 4.3, significantly higher than that of aspartic acid.
• In some proteins, due to a vitamin K dependent carboxylase, some
glutamic acids will be dicarboxylic acids, referred to as γ
carboxyglutamic acid, that form tight binding sites for calcium ion.
• Arginine, an essential amino acid, has a positively charged guanidino
group. Arginine is well designed to bind the phosphate anion, and is
often found in the active centers of proteins that bind phosphorylated
substrates.
• As a cation, arginine, as well as lysine, plays a role in maintaining the
overall charge balance of a protein.
• There are 6 codons in the genetic code for arginine, yet, although this
large a number of codons is normally associated with a high frequency
of the particular amino acid in proteins, arginine is one of the least
frequent amino acids.
• The discrepancy between the frequency of the amino acid in proteins
and the number of codons is greater for arginine than for any other
amino acid.
Non-polar amino acids
• They have equal number of amino and carboxyl
groups and are neutral.
• These amino acids are hydrophobic and have no
charge on the 'R' group. The amino acids in this
group are alanine, valine, leucine, isoleucine,
phenyl alanine, glycine, tryptophan, methionine
and proline.
Non-polar amino acids
Polar amino acids with no charge
• These amino acids do not have any charge on
the 'R' group. These amino acids participate in
hydrogen bonding of protein structure.
• The amino acids in this group are - serine,
threonine, tyrosine, cysteine, glutamine and
aspargine
Polar amino acids with no charge
Polar amino acids with positive charge
• Polar amino acids with positive charge have
more amino groups as compared to carboxyl
groups making it basic.
• The amino acids, which have positive charge on
the 'R' group are placed in this category. They
are lysine, arginine and histidine.
• Polar amino acids with negative charge
• Polar amino acids with negative charge have
more carboxyl groups than amino groups making
them acidic.
• The amino acids, which have negative charge on
the 'R' group are placed in this category. They are
called as dicarboxylic mono-amino acids. They
are aspartic acid and glutamic acid.
Types of Amino acids
1. Aromatic group
2. Aliphatic group
3. Sulphur Containing amino acids
4. Hydrophylic amino acids
• Aromatic amino acids are normally hydrophobic and includes
phenylalanine, tyrosine and tryptophan.
•
Aliphatic amino acids are basically hydrophobic and an be
located in core of protein. glycine ,valine, alanine, leucine,
proline and isoleucine are aliphatic amino acids.
• sulphur containing amino acids include sulphur atom and
cysteine and methionine are the examples.
• Hydrophilc amino acids are further categorized as
acidic ,neutral and basic amino acids. Acidic amino
acids are highly polar and are always negatively
charged. Aspartate and glutamate are the examples.
• Basic amino acids contains side chains that are
positively charged . lysine,arginine and histidine are the
examples.
• Neutral amino acids are polar in nature and serine
,threonine, asparagine and glutamine are the
examples.
Introduction
• Proteins are the machines that drive cells and,
ultimately, organisms.
Proteins are composed of individual units called amino
acids. Amino acids all share a similar structure. The
difference between them is the so-called "R" group. The
"R" group is the cluster of atoms that give an amino acid its
particular characteristics.
• Proteins are not linear molecules as suggested
when we write out a "string" of amino acid
sequence, -Lys-Ala-Pro-Met-Gly- etc., for example.
• Rather, this "string" folds into an intricate threedimensional structure that is unique to each
protein.
• It is this three-dimensional structure that allows
proteins to function.
• Thus in order to understand the details of protein
function, one must understand protein structure.
Formation of Peptide Bond in AA
↓
→
H
O
↓
2
Peptide Bond
↓
Amino Acids
• The amino acid residues of proteins are defined by an amino group and
a carboxyl group connected to an alpha carbon to which is attached a
hydrogen and a side chain group R.
• The smallest amino acid, glycine, has a hydrogen atom in place of a side
chain.
• All other amino acids have distinctive R groups. Because the alpha
carbon of the other amino acids have four different constituents, the
alpha carbon atom is an asymmetric center and most naturally
occurring amino acids are in the L form.
(S)-Alanine (left) and (R)-alanine (right) in
zwitterionic form at neutral pH
(S)- Alanine (left)
(R)-alanine (Right)
• Any number of amino acids can be joined
together to form peptides of any length.
• Small peptides (containing less than a couple of
dozen amino acids) are sometimes called
oligopeptides. Longer peptides are many times
called polypeptides.
• Notice that peptides have a "polarity"; each
peptide has only one free a-amino group (on the
amino-terminal residue) and one free (nonsidechain) carboxyl group (on the carboxyterminal residue)
• Amino acids fall into several naturally occurring groups
including hydrophobic, hydrophilic, charged, basic, acidic,
polar but uncharged, small polar, small hydrophobic, large
hydrophobic, aromatic, beta-branched, sulfur containing
etc.
• Hydrophobic amino acids, sometimes called non-polar
amino acids, reside primarily on the interior of the
protein.
• Hydrophilic amino acids, sometimes called polar amino
acids, reside primarily on the exterior of the protein.
• Many amino acids will fall into more than one group since
each amino acid side chain has several properties.
Forces determining protein structure
• Several covalent and non-covalent
determine protein structure.
1) van der Waals interactions
2) Hydrophobic force
3) Electrostatic forces
4) Dipole moments
5) Hydrogen bonds
6) Covalent bond
forces
1. Van der Waals interactions
• interactions between immediately adjacent atoms:
These non-covalent forces result from the attraction of
one atoms nucleus for the electrons of another atom in a
non-covalent form (no sharing of orbitals).
• These forces are much weaker than covalent interactions
and the interaction distances are much longer than
covalent bonds and much shorter than the other noncovalent interactions.
• Van der Waals interactions are non-directional and very
weak. However, significant energy of stabilization can be
obtained in the central hydrophobic core of proteins by
the additive effect of many such interactions.
2.Hydrophobic force
• The hydrophobic force is really a negative non-covalent
force.
• The presence of hydrophobic side chains in aqueous
solution induces the formation of structured water
(clathrate cages of water molecule form, like miniature ice
crystals about the hydrophobic side chains).
• The hydrophobic force is one of the largest determinants
of protein structure. Most secondary structural elements
we will discuss have an amphipathic nature, one
hydrophobic side and one hydrophilic surface of the
protein.
3.Electrostatic forces
• The attraction of oppositely charged side chains can form saltbridges that stabilize secondary and tertiary structures.
• The electrostatic force is quite strong, falling off as the square of
the distance between the charged atoms.
• It also depends heavily on the dielectric constant of the
medium in which the protein is dissolved.
•
It is strongest in a vacuum and 80 fold weaker in water and
weaker still at elevated salt solutions.
• Water and ions can shield electrostatic interactions reducing
both their strength and distance over which they operate.
4.Dipole moments
• Dipole moments are caused by pairs of charges separated by a larger
distance than permitting a salt- or ion bridge.
• The dipole moment gives rise to an electric field along the entire
length of a structural element.
• Dipole moments are often used by proteins to attract and position
charged substrates and products.
•
The peptide chain naturally has a dipole moment because the Nterminus carries about 1/2 a positive charge and the C-terminus carries
about 1/2 unit of negative charge.
• The alpha helix is known to carry a partial negative charge at its Cterminus and a positive charge at its N-terminus.
• In order to help neutralize this charge distribution, alpha helices often
have acidic residues near their N-terminus and a basic residue near
their C-terminus.
5. Hydrogen bonds
• Hydrogen bonds occur when a pair of nucleophilic atoms such as oxygen
and nitrogen share a hydrogen between them.
• The hydrogen may be covalently attached to either nucleophilic atom (the
H-bond donor) and shared with the other atom (the H-bond receptor).
• H-bonds are directional and their strength deteriorates dramatically as the
angle changes.
• Hydrogen bonds do not, in general, contribute to the net stabilization
energy of proteins because the same groups that hydrogen bond to each
other in a native protein structure, can hydrogen bond to water in the
denatured state.
• However, hydrogen bonds are extremely important because of their
directionality, they can control and limit the geometry of the interactions
between side-chains.
• This is shown most dramatically in patterns of hydrogen bonding between
the carboxyl groups and the amino groups in the peptide backbone that
give rise to alpha helix and beta strand conformations.
6. Covalent bond
• The major properties of the covalent bonds hold
proteins together are their bond distances and
bond angles.
• In particular, the bond angles between two
adjacent bonds on either side of a single atom,
or the dihedral angles between three contiguous
bonds and two atoms control the geometry of
the protein folding.
Primary Structures- Protein
• Primary structure refers to the "linear" sequence
of amino acids.
• Primary structure is sometimes called the
"covalent structure" of proteins because, with the
exception of disulfide bonds, all of the covalent
bonding within proteins defines the primary
structure.
• Generally, peptides are small 10 or 20 residues;
polypeptides might range up to 50 or 60 residues,
• Small peptides (containing less than a couple of dozen
amino acids) are sometimes called oligopeptides.
Longer peptides are many times called polypeptides.
• each peptide has only one free a-amino group (on the aminoterminal residue) and one free (non-sidechain) carboxyl group
(on the carboxy-terminal residue):
Primary Structure of Protein
Secondary Structures (SS)
• Secondary structure is the initial folding
pattern (periodic repeats) of the linear
polypeptide. There are 2 main types of
secondary structure: α- helix and β-sheet
Secondary structures are stabilized by
hydrogen bonds.
The α-helix
• The α-helix is right-handed or clock-wise (for L
isoforms left-handed helix is not viable due to
steric hindrance) Each turn has 3.6 aa residues and
is 5.4 A° high.
• The helix is stabilized by H-bonds between –N-H
and –C=O groups of every 4th amino acid.
• α-helices can wind around each other to form
‘coiled coils’ that are extremely stable and found in
fibrous structural proteins such as keratin, myosin
(muscle fibers) etc
The α-helix Structure
Five different kinds of constraints affect the stability of an a
helix:
• The electrostatic repulsion (or attraction) between
successive amino acid residues with charged R groups.
• The bulkiness of adjacent R groups.
• The interactions between amino acid side chains spaced
three (or four) residues apart.
• The occurrence of Pro and Gly residues.
• The interaction between amino acid residues at the ends
of the helical segment and the electric dipole inherent to
the a helix.
β-Pleated Sheet
• Extended stretches of 5 or more aa are called β- strands.
β-strands organized next to each other make β-sheets.
• If adjacent strands are oriented in the same direction (Nend to C-end), it is a parallel β-sheet, if adjacent strands
run opposite to each other, it is an antiparallel β-sheet.
• There can also be mixed β-sheets. The H-bonding
pattern varies depending on type of sheet.
• β-sheets are usually twisted rather than flat.
• Fatty acid binding proteins are made almost entirely of
β-sheets
β-Pleated Sheet
Tertiary Structure
• 3D folding or ‘bundling up’ of the protein is the tertiary
structure of the proteins.
• Non-polar residues are buried inside, polar residues are
exposed outwards to aqueous environment.
• Many proteins are organized into multiple ‘domains’ which
are compact globular units that are connected by a flexible
segment of the polypeptide.
• Each domain contributes a specific function to the protein.
Different proteins may share similar domain structures,
eg: kinase-, cysteine-rich-, globin-domains.
Tertiary Structures
The protein then can compact and twist on itself to
form a mass called it’s Tertiary Structure
5 kinds of bonds stabilize tertiary
structure
•
•
•
•
•
H-bonds,
van der waals interactions,
hydrophobic interactions,
ionic interactions and
disulphide linkages
In disulphide linkages, the SH groups of two neighboring
cysteines form a –S-S- bond called as a disulphide linkage.
It is a covalent bond, but readily cleaved by reducing agents that
supply the protons to form the SH groups again.
Quaternary Structure.
• Quaternary structure is a larger assembly of several
protein molecules or polypeptide chains, usually
called subunits in this context.
• The quaternary structure is stabilized by the same
non-covalent interactions and disulfide bonds as
the tertiary structure.
• Complexes of two or more polypeptides (i.e.
multiple subunits) are called multimers.
• Specifically it would be called a dimer if it contains two
subunits, a trimer if it contains three subunits, and a
tetramer if it contains four subunits.
• The subunits are frequently related to one another by
symmetry operations, such as a 2-fold axis in a dimer.
• Multimers made up of identical subunits are referred to
with a prefix of different subunits are referred to
"hetero-" (e.g. a heterotetramer, such as the two alpha
and two beta chains of hemoglobin).
• Many proteins do not have the quaternary structure
and function as monomers.
Protein Structures Overview
Several Proteins then can combine and form a
protein’s Quaternary Structure
Functions of Protein
What does Protein Do?
• Protein has a large number of important functions in the
human body—and in fact, the human body is about 45%
protein. It’s an essential macromolecule without which
our bodies would be unable to repair, regulate, or
protect themselves.
• Proteins are, in effect, the main actioners in cells and in
an entire organism. Without proteins the most basic
functions of life could not be carried out. Respiration,
for example, requires muscle contractions, and muscle
contractions require proteins.
Protein has a range of essential functions in the body,
including the following:
• Required for building and repair of body tissues
(including muscle)
• Enzymes, hormones, and many immune molecules
are proteins
• Essential body processes such as water balancing,
nutrient transport, and muscle contractions require
protein to function.
• Protein is a source of energy.
• Protein helps keep skin, hair, and nails healthy.
• Protein, like most other essential nutrients, is
absolutely crucial for overall good health.
Proteins as Enzymes
• The function of proteins as enzymes is perhaps their bestknown function. Enzymes are catalysts—they initiate a
reaction between themselves and another protein,
working on the molecule to change it in some way.
• The enzyme, however, is itself unchanged at the end of
the reaction.
• Enzymes are responsible for catalyzing reactions in
processes such as metabolism, DNA replication, and
digestion.
• In fact, enzymes are known to be involved in some 4,000
bodily reactions.
Proteins in Cellular Signaling and Molecular Transport
• Cells signal one another for an enormous variety of reasons,
the most basic of which is simply to coordinate cellular
activities. Signaling is how cells communicate with one
another, allowing such essential processes as the contraction
of the heart muscle to take place.
• Proteins are important in these processes due to their ability
to bind other molecules—a protein produced by one cell may
bind to a molecule produced by another, thus providing a
chemical signal which allows the cells to provide information
about their state. Proteins are also involved in molecular
transport.
• A prime example of this is the protein called hemoglobin,
which binds iron molecules and transports them in the blood
from the lungs to organs and tissues throughout the body.
• Structural proteins are those which confer strength and
rigidity to biological components which would otherwise
be unable to support themselves.
• Structural proteins tend to have very specific shapes—
long, thin fibers or other shapes which, when allowed to
form polymers, provide strength and support.
• Structural proteins are essential components of collagen,
cartilage, nails and hair, feathers, hooves, and other such
components.
• Structural proteins are also essential components of
muscles, and are necessary to generate the force which
allows
muscles
to
contract
and
move.
• Though enzymes exhibit great degrees of
specificity, cofactors may serve many
apoenzymes.
• For
example,
nicotinamide
adenine
dinucleotide (NAD) is a coenzyme for a great
number of dehydrogenase reactions in which
it acts as a hydrogen acceptor.
• Among them are the alcohol dehydrogenase,
malate
dehydrogenase
and
lactate
dehydrogenase reactions.
Enzymes can be classified by the kind of chemical reaction catalyzed
• Addition or removal of water
– Hydrolases - these include esterases, carbohydrases, nucleases, deaminases,
amidases, and proteases
– Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase
• Transfer of electrons
– Oxidases
– Dehydrogenases
• Transfer of a radical
– Transglycosidases - of monosaccharides
– Transphosphorylases and phosphomutases - of a phosphate group
– Transaminases - of amino group
– Transmethylases - of a methyl group
– Transacetylases - of an acetyl group
• Splitting or forming a C-C bond
– Desmolases
• Changing geometry or structure of a molecule
– Isomerases
• Joining two molecules through hydrolysis of pyrophosphate bond in ATP or other triphosphate
– Ligases
Enzyme Kinetics: Basic Enzyme Reactions
• Enzymes are catalysts and increase the speed of
a chemical reaction without themselves
undergoing any permanent chemical change.
They are neither used up in the reaction nor do
they appear as reaction products.
• The basic enzymatic reaction can be represented
as follows
• where E represents the enzyme catalyzing the
reaction, S the substrate, the substance being
changed, and P the product of the reaction.
Enzyme Kinetics: The Enzyme Substrate Complex
• A theory to explain the catalytic action of
enzymes was proposed by the Swedish chemist
Savante Arrhenius in 1888.
• He proposed that the substrate and enzyme
formed some intermediate substance which is
known as the enzyme substrate complex. The
reaction can be represented as:
• The existence of an intermediate enzymesubstrate complex has been demonstrated in
the laboratory, for example, using catalase and
a hydrogen peroxide derivative.
• This experimental evidence indicates that the
enzyme first unites in some way with the
substrate and then returns to its original form
after the reaction is concluded.
The Michaelis-Menten equation is a quantitative
description of the relationship among the rate of an
enzyme- catalyzed reaction [v1], the concentration of
substrate [S] and two constants, Vmax and Km (which are
set by the particular equation).
The symbols used in the Michaelis-Menten equation refer
to the reaction rate [v1], maximum reaction rate (Vmax),
substrate concentration [S] and the Michaelis-Menten
constant (Km).
Factors Affecting Enzyme Activity
• Knowledge of basic enzyme kinetic theory is important
in enzyme analysis in order both to understand the
basic enzymatic mechanism and to select a method for
enzyme analysis.
• The conditions selected to measure the activity of an
enzyme would not be the same as those selected to
measure the concentration of its substrate.
• Several factors affect the rate at which enzymatic
reactions proceed - temperature, pH, enzyme
concentration, substrate concentration, and the
presence of any inhibitors or activators.
Enzyme Concentration
• In order to study the effect of increasing the enzyme
concentration upon the reaction rate, the substrate must be
present in an excess amount; i.e., the reaction must be
independent of the substrate concentration.
• Any change in the amount of product formed over a specified
period of time will be dependent upon the level of enzyme
present. Graphically this can be represented as:
• The amount of enzyme present in a reaction is
measured by the activity it catalyzes.
• The
relationship
between
activity
and
concentration is affected by many factors such as
temperature, pH, etc.
• An enzyme assay must be designed so that the
observed activity is proportional to the amount of
enzyme present in order that the enzyme
concentration is the only limiting factor.
• It is satisfied only when the reaction is zero order.
Substrate Concentration
• If the amount of the enzyme is kept constant
and the substrate concentration is then
gradually increased, the reaction velocity will
increase until it reaches a maximum. After this
point, increases in substrate concentration will
not increase the velocity (∆A / ∆T).
• It is theorized that when this maximum
velocity had been reached, all of the available
enzyme has been converted to ES, the enzyme
substrate complex.
• Michaelis constants have been determined for many of the
commonly used enzymes. The size of Km tells us several
things about a particular enzyme.
• A small Km indicates that the enzyme requires only a small
amount of substrate to become saturated. Hence, the
maximum velocity is reached at relatively low substrate
concentrations.
• A large Km indicates the need for high substrate
concentrations to achieve maximum reaction velocity.
• The substrate with the lowest Km upon which the enzyme
acts as a catalyst is frequently assumed to be enzyme's
natural substrate, though this is not true for all enzymes.
Effects of Inhibitors on Enzyme Activity
• Enzyme inhibitors are substances which alter
the catalytic action of the enzyme and
consequently slow down, or in some cases,
stop catalysis. There are three common types
of enzyme inhibition - competitive, noncompetitive and substrate inhibition.
• Most
theories
concerning
inhibition
mechanisms are based on the existence of the
enzyme-substrate complex ES. As mentioned
earlier, the existence of temporary ES
structures has been verified in the laboratory.
Temperature Effects
Like most chemical reactions, the rate of an enzyme-catalyzed
reaction increases as the temperature is raised. A ten degree
Centigrade rise in temperature will increase the activity of most
enzymes by 50 to 100%. Variations in reaction temperature as
small as 1 or 2 degrees may introduce changes of 10 to 20% in the
results. In the case of enzymatic reactions, this is complicated by
the fact that many enzymes are adversely affected by high
temperatures.
• The reaction rate increases with temperature to
a maximum level, then abruptly declines with
further increase of temperature. Because most
animal enzymes rapidly become denatured at
temperatures above 40°C, most enzyme
determinations are carried out somewhat below
that temperature.
• Over a period of time, enzymes will be
deactivated at even moderate temperatures.
Storage of enzymes at 5°C or below is generally
the most suitable. Some enzymes lose their
activity when frozen.
Effects of pH
• Enzymes are affected by changes in pH. The most
favorable pH value - the point where the enzyme
is most active - is known as the optimum pH.
• Extremely high or low pH values generally result
in complete loss of activity for most enzymes. pH
is also a factor in the stability of enzymes. As
with activity, for each enzyme there is also a
region of pH optimal stability.
Enzyme
pH Optimum
Lipase (pancreas)
8.0
Lipase (stomach)
4.0 - 5.0
Lipase (castor oil)
4.7
Pepsin
1.5 - 1.6
Trypsin
7.8 - 8.7
Urease
7.0
Invertase
4.5
Maltase
6.1 - 6.8
Amylase (pancreas)
6.7 - 7.0
Amylase (malt)
4.6 - 5.2
Catalase
7.0
• In addition to temperature and pH there are
other factors, such as ionic strength, which can
affect the enzymatic reaction. Each of these
physical and chemical parameters must be
considered and optimized in order for an
enzymatic reaction to be accurate and
reproducible.
Enzyme Function
• In simple terms, an enzyme functions by binding
to one or more of the reactants in a reaction.
• The reactants that bind to the enzyme are known
as the substrates of the enzyme.
• The exact location on the enzyme where substrate
binding takes place is called the active site of the
enzyme.
• The shape of the active site just fits the shape of
the substrate, somewhat like a lock fits a key.
• In this way only the correct substrate binds to the
enzyme.
• Once the substrate or substrates are bound to
the enzyme, the enzyme can promote the
desired reaction in some particular way.
• What that way is depends on the nature of
the reaction and the nature of the enzyme. An
enzyme may hold two substrate molecules in
precisely the orientation needed for the
reaction to occur.
• Or binding to the enzyme may weaken a bond
in a substrate molecule that must be broken in
the course of the reaction, thus increasing the
rate at which the reaction can occur.
• An enzyme may also couple two different
reactions.
• Coupling an exothermic reaction with an
endothermic one allows the enzyme to use the
energy released by the exothermic reaction to
drive the endothermic reaction.
• In fact, a large variety of enzymes couple many
different endothermic reactions to the exothermic
reaction in which ATP is converted by hydrolysis to
ADP.
• In this way, ATP serves as the molecular fuel that
powers most of the energy-requiring processes of
living things.