Doc Brown's Advanced A Level
Organic Chemistry Revision Notes - Help in Revising Advanced Organic Chemistry
PART 14 ORGANIC ISOMERISM
and Stereochemistry Revision Notes
14.4 Notes on protein
structure, enzyme structure and function
studies of structure, formation, properties and consequences
brown - comments - query?
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All my advanced A level
isomerism and stereochemistry notes
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Stereoisomerism, aspects of stereochemistry contd.
this page 14.4: 2b.6 sub-index:
notes on protein/enzyme structure and function
enzyme function *
protein structure -
structure of silk and wool
enzyme co-factors/co-enzymes *
enzyme inhibition *
to E/Z (geometric) and R/S (optical) isomerism :
amino acids *
alanine synthesis *
lactic acid synthesis
nucleophilic substitution of halogenoalkanes* 14.6
chemistry and autosynthesis *
14.4 Case studies - Extra
notes on protein/enzyme structure and function
to section 2b.6 and glimpse of the world of biochemistry!
E. coli bacteria contain about over
5000 different compounds, most of which are macromolecules, including over
3000 proteins/polypeptides (over 100000 in humans) and about 1000
different nucleic acids in the form of RNA and DNA. All bar a
small % of proteins are built from the same 'pool' of 20 alpha amino acids
in the cytoplasm of cells, despite the fact that 150 other amino acids
have been found in living organisms too.
Over 2000 enzymes are known and several hundred proteins have been
crystallised and their detailed 3D structure determined by x-ray
crystallography. It has estimated that a polypeptide of Mr
34000, built from 12 different amino acids can have 10300
different sequences, now that's what you call isomerism!
All of the
DNA and RNA nucleic acids are built from just 8 nucleotides
(phosphate-sugar-base 'monomers') and are all constructed with the help of
enzymes. All the thousands of different reactions keeping an
organism going are specific and efficient from cell division to
respiration. So, to finish where we started, what can be the cause
of a very nasty, and potentially fatal, tummy bug, is a very different
chemical world from refluxing a halogenoalkane with sodium hydroxide to
make an alcohol in the laboratory, but its a bit too much to expect us to
intellectually marvel at the wonders of biochemistry when we are feeling
Enzymes are proteins,
which are themselves built up of amino acid residue sequences (primary structure).
The specificity of enzymes is due to their structure, from a specific
amino acid sequence to the full 3-dimensional
globular structure, which in turn controls the nature of the chemical
bonds formed, or interaction via intermolecular forces, with the
So biosynthesis in
living systems, can operate through enzymes in a
stereospecific way. The diagram of the 'key and lock' mechanism is
obviously presented in 2D, but in reality, it is a 3D situation and
e.g. only one optical isomers might be able to 'dock' successfully or only
one optical isomer is formed. The diagram
shows a molecule being broken down e.g. protein digestion to give amino
acids, BUT you could also think of the sequence working from right to left
i.e. producing protein from amino acids via a different enzyme system and
the purple and brown 'shapes' represent substrate molecules.
So the enzymes activity depends
on its tertiary and quaternary structure and the specific part of an enzyme
where a chemical change takes place is called the active site. This
has a specific 3D molecular shape which allows the compact 'docking
and holding' (via temporary bonds or intermolecular forces) of the
substrate molecule and providing a lower energy pathway to break/make
bonds of the substrate/product molecules. More details on
the background to understanding the specificity, pH and temperature
sensitivity and inhibition of enzymes are given
also: GCSE notes
rates of reaction and uses and
notes on enzyme kinetics
of proteins in living organisms cannot be over-emphasised e.g. their
existence as the hormone insulin and enzyme molecules ('biological
catalysts') or in muscle/membrane/hair structure and even the oxygen
transporter haemoglobin which is a protein-Fe-O2 complex when
red!, and now the growing area of biotechnology to manufacture a whole
range of chemical products. In the form of
enzymes, they are highly reactive and selective towards substrate molecules, catalyzing a
multitude of specific chemical reactions often in complex multi-stage
specificity and sensitivity of enzymes:
The structure of
enzyme-protein molecules is described in various ways below and the
very complex and specific nature of each enzyme structure explains why
they will only interact with specific substrate molecules. The
descriptions below also explain an enzymes sensitivity to increased
temperature or pH changes.
Please remember, they only increase the
kinetics of the reactions, and, although they decrease the activation
energy, they CANNOT change the enthalpy of a reaction or the % yield if
an equilibrium is formed.
An example of
the stereospecificity of enzymes is the action of the fumarase in converting
trans-butenedioic acid (fumaric acid) into 2-hydroxybutanedioic
acid (malic acid).
HOOC-CH=CH-COOH + H2O ==
fumarase ==> HOOC-*CH(OH)-CH2-COOH
acid has a chiral or asymmetric carbon (*C),
so two optical isomers (enantiomers) are possible, BUT only L
enantiomer is formed, and non of the D enantiomer.
Not only is the
product a stereospecific optical isomer, the reactant is itself
a stereospecific geometrical isomer!
TOP OF PAGE
The importance of proteins
Proteins are extremely important
molecules in living systems and very little biochemistry happens without
them being involved.
important biological molecules e.g. in tissue structure of muscles and
skin, they act as enzymes, hormones, antibodies of the immune system and
proteins aid and control the transport of substances in and out of cells
The structure of proteins
Proteins can be divided into two
classes depending on their large scale structure
(i) Fibrous proteins contain long chains of
polypeptides which often occur in bundles e.g. keratin and are
usually insoluble in water - feathers, shells and horns are of no
use if they dissolve in water
They are structural fibrous proteins are made up
of elongated or fibrous polypeptide chains which form
filamentous and sheet-like structures
proteins consist of many types including keratin, collagen,
elastin, and fibrin.
Collagen is the most
abundant of these proteins which exists in vertebrate connective
tissue including tendon, cartilage, and bone.
(ii) Globular proteins are folded into a
roughly spherical shape and can be soluble in water.
Globular proteins can act as:
Enzymes, by catalyzing organic reactions taking place in the
organism in mild conditions and with a great specificity.
Messengers, by transmitting messages to regulate biological
processes. This function is done by hormones, i.e. insulin which
is made up of 51 amino acids.
other molecules through membranes.
roles are also performed by globular proteins rather than
(iii) Membrane proteins
Membrane proteins are a permanent integral part
of a cell membrane and perform a variety of functions vital to
the survival of organisms e.g.
proteins relay signals between the cell's internal and external
Transport proteins move molecules
and ions across the membrane.
may have many activities.
Cell adhesion molecules
allow cells to identify each other and interact e.g. proteins
involved in immune response
The fine details of protein structure are now
The order of the amino acids in the polypeptide
chain (the sequence of residues).
synthesis-formation is illustrated in a later section.
structure sequence only involves covalent bonding i.e. the
condensation reaction involves the formation of the polypeptide
or polyamide linkage -NH-CO- to give a natural condensation
polymer (since a small molecule, water, is eliminated in the process).
A generalised diagram of a section of a polypeptide or
section of a protein molecule, but only in terms of amino acid residues.
A sequence of 7
amino acid residues is shown. In biochemistry or molecular biology, a
residue refers to a single unit that makes up a polymer, such as an
amino acid in a polypeptide or protein.
peptide linkage formed by loss of H from the H2N
group and OH from the COOH group i.e. the loss of H2O between two amino
acids to give the HN-C=O
Also, note for the peptide link, the ~120o bond angles for C-C=O,
C-C-N and O=C-N giving a trigonal planar arrangement of bonds around
the carbon atom of the peptide linkage HN-CO.
In this diagram the 7 amino residues are made specific to
give the sequence Arg-Phe-Asp-Glu-Ser-Cys-Ala,
note the convenience of using the three letter code abbreviations!
There is one very important aspect which is not
appreciated from the diagram.
I've dropped down all the R
groups in one direction, but there is free rotation about the C-N, C-C
linking bonds in the polymer chain.
So, via the differing
R groups, the polypeptide/protein molecule can adopt a huge variety of complex 3D
shapes as in enzymes (e.g. diagram on right).
The 3rd version of this diagram is to
show you how to read the sequence of amino acid residues in a polypeptide
and identify the original amino acid. Each residue is sectioned off by the
diagonal purple lines.
e.g. the 3rd one in the sequence is Asp aspartic acid.
The residue structure is HN-CH(CH2-COOH)-CO,
so the equivalent of H2O is added, to get to the original
amino acid structure is H2N-CH(CH2-COOH)-COOH
The coiling or folding of the polypeptide chain into an
or the formation of a beta-pleated
sheet of protein is stabilised by
hydrogen bonding (see
Alpha-helix form of proteins
In the alpha-helix form the
polypeptides are coiled in a spiral whose structure is held together
by intramolecular hydrogen bonds - these occur every amino acid four
residues ('4 units' derived from the polymerised amino acids).
Intramolecular means 'within the same molecule' i.e. in this
part of polypeptide>C=Oδ-llllδ+H-N<another
part of same polypeptide
You also get ionic bonds between basic amino
groups and carboxylic acids.
Myoglobin is a protein with an alpha-helical
Beta-pleated sheet form of
In these protein structures the
polypeptide chains are mostly fully extended and held by
bonds to other polypeptide chains (see
structure of silk and wool).
Intermolecular means 'between molecules'
i.e. in this context:
The sheets take up a 'corrugated' formation that
make a very strong fibre because of the extensive regular
intermolecular hydrogen bonds between the polypeptide chains.
This is the full 3D shape
(conformation) of the protein is produced by the holding
together the secondary structure (alpha-helix or beta-sheet) into a full
structure is stabilised by
four types of inter-molecular
force or chemical bonding which can be between >C=O and
H-N groups of molecular/ionic features in the variable side chain 'R'
Enzymes, for example, might
incorporate both alpha helix and beta sheet sections of polypeptide
In some cases, single protein molecules can join
together to give a larger unit e.g. , insulin can
exist as a monomer, dimer (insulin)2 or a
The quaternary structure of dimers,
trimers etc. is often held together by complexing
with a metal ion.
In the insulin hexamer, the insulin acts as a ligand
forming dative covalent bonds with a Zn2+ ion or
intermolecular forces e.g. hydrogen bonding in
holds 4 components together.
Four types of
interaction are important in holding together the secondary and
dipole-induced dipole (Van der Waals) intermolecular attractive forces,
the main contribution from non-polar side chains of the R group.
(ii) Permanent dipole-permanent
dipole intermolecular forces, often in due to the strongest type in the form
of hydrogen bonding
bonding - ionisable side chain groups e.g. between
(iv) Covalent bonding
e.g. disulfide bridges
Examples are described in detail below AND comments added on how these
interactions may be changed to affect enzyme function and efficiency
with respect to increase in temperature or pH change.
also a discussion of enzyme kinetics - rates of reaction
TOP OF PAGE
structure of proteins and denaturing (and
of secondary or tertiary structures are described below and references to
dipole-induced dipole forces between polypeptide chains
Non-polar Van der Waals forces,
hydrophobic interactions in the presence of water.
arise from the random behaviour of the electron fields in
atoms producing minute transient dipoles
which in turn induce δ+moleculeδ-
dipoles in neighbouring molecules. They are the
weakest of the intermolecular forces, but here we are not talking
molecule-molecule interaction (inter-molecular forces), but within
one big molecule we are talking molecular section-section interaction, and
the same argument
applies for 2. below.
The centres of protein molecules often have
amino acids with hydrocarbon sections (e.g. the side chains of
Leucine, isoleucine and phenylalanine shown below). These
non-polar side chains in the polypeptide do not affect hydrogen
bonding of the protein with surrounding water molecules or polar
The residues (Leu,
Ile etc.) are the parts of the original amino acids which are used to form the
protein chain i.e. after loss of H2O from -COOH + H2N-
to give linked adjacent amino acid residues.
leucine, 2-amino-4-methylpentanoic acid,
isoleucine, 2-amino-3-methylpentanoic acid,
phenylalanine, 2-amino3-phenylpropanoic acid,
the -NH2 and -COOH on the right are used in
forming the -NH-CO- peptide linkage, so concentrate on
the left-hand side i.e. the 'side chain', and do the same
for the other examples below.
dipole-induced dipole force interactions will be the first to be
weakened and disrupted on increase in temperature and
beginning the thermal 'denaturing' or 'degradation' of
the tertiary 3D structure of an
protein, including enzymes. Once the conformation is
changed the enzyme is catalytically inactive.
Note: Most enzymes are
'globular' in shape and the hydrophobic non-polar side-chain
groupings (above in section 1.) tend to align alongside each other
towards the centre of the 3D conformation.
If not involved in holding
the tertiary structure, the hydrophilic polar side-chain groupings
(below in section 2.) tend to be directed towards the outer 'surface' to
interface with the aqueous media the enzyme operates in.
So each type of
intermolecular force does not interfere with the others function.
TOP OF PAGE
2. Polar bonds
and hydrogen bonding between polypeptide
Polar permanent dipole-permanent
dipole intermolecular forces, hydrophilic interactions in the presence
dipoles arise when two atoms of a bond in a molecule have different
electronegativities. Electronegativity is a measure of the electron
attracting power of an atom in a covalent bond situation, and in polar
bond, the more electronegative element attracts more of the bonding
electron cloud to give an asymmetric distribution i.e. a permanent
dipole. These dipoles occur between the carbonyl
group δ+C=Oδ- and
the secondary amide group
of one peptide linkage (-NH-CO-) and the peptide linkage in the same
chain or another chain. There are also H-bond interactions between proteins and water in an
aqueous media. The hydrogen bond,,
is the strongest of the intermolecular forces
arising from the big electronegativity difference
between O/N and H in these cases (electronegativity of O/N >
H). It is NOT a chemical bond
like an ionic or covalent bond but it is the strongest of the permanent
dipole - permanent dipole interactions between molecules OR sections of a
molecule, i.e. hydrogen bonding is the strongest of the intermolecular
Hydrogen bonding between an alcohol group in the side chain of an amino
acid residue and water surrounding a protein molecule.
Hydrogen bonding between an alcohol group in the side chain of an amino
acid residue and an amine group in a side-chain.
Hydrogen bonding between a carbonyl group and water with a protein in an
Hydrogen bonding between the carbonyl group of one peptide linkage and the
secondary amide group of another polypeptide linkage.
The link could be
between 'folds' in the same polypeptide chain e.g. the alpha-helix form (H-bond
every 3-4 residues holding the coils of the helix in place, see
diagram) OR between two separate
polypeptide chains held together e.g. beta-sheets of protein material,
shown schematically below.
chains held together to produce the '2D' arrangement of a beta-sheet of
protein. The structural formula, the bond polarities and the hydrogen
bonds are shown. The variable R side-chain grouping is alternately above
and below the '2D planarity' of the sheet which extends in the dimensions
of the screen/paper printout (left-right covalent bonding, up-down
hydrogen-bonding). The sheets are quite strong and you would find this
sort of arrangement in collagen or muscle tissue.
simpler diagram of the hydrogen bonding via the skeletal formula (the H's
to the N must be shown in the skeletal formula to show the hydrogen
The ....δ+C=Oδ-....δ+H-Nδ-.... intermolecular force
interaction is the most important hydrogen bond that holds together the secondary
structure of proteins to give the sheet and helical
structures AND about 50% of the hydrogen bonds that hold
together the single helix structure of RNA molecules and the double
helix structure of DNA molecules.
permanent dipole interactions
can also occur between
polar side-chain groups as well as the polypeptide
-NH- and -CO- links (see
alpha-helix below and beta-sheet above) and usually involve
between neighbouring highly polar groups. For example,
groups in the side chains of serine, threonine, asparagine and glutamine.
look for the possibility of
serine, 2-amino-3-hydroxypropanoic acid
one exemplar hydrogen bond,
it could involve any of the amino acid
residues described below,
H-O in another part of the polypeptide
on a side chain.
Covalent bonds connect one residue via the CO-NH link to the next amino acid
polypeptide linkage between two residues is NH-CO.
threonine, 2-amino-3-hydroxybutanoic acid
on a side chain
H2N or C=O
on a side chain
H2N or C=O
on a side chain
aspartic acid, 2-aminobutanedioic acid,
OH or C=O
on a side chain
lysine, 2,6-diaminohexanoic acid,
on a side
What can cause
the denaturing of a protein?
If a protein is heated or is subjected to a
very high or very low pH, the intermolecular bonding, particularly
hydrogen bonds, are broken which disrupts the intermolecular
bonding holding the secondary or tertiary structure together - the
protein is then described as denatured and cannot function as
its original form i.e. enzyme activity reduced or terminated - the
active site is adversely affected by any 3D change in its conformation
like the transient dipole-induced dipole forces, these
stronger, BUT still relatively weak hydrogen bonding inter-molecular forces, will be
increasingly weakened/broken as the temperature is raised.
The 'effective' 3D protein structure
(conformation) is 'denatured' as more hydrogen
bonds get broken. Both secondary and tertiary structure
is destroyed but not the strong covalent bonds of the
The possible effect of pH change
via acids and alkalis (other 'denaturing agent') on hydrogen
bonding and ionic bonds, is
conveniently described in section 3. below, but
please remember the above hydrogen bonds are NOT chemical bonds!
The 'schematic' diagram below
shows the denaturing effect on the secondary
structure of proteins, particularly by the disruption of hydrogen bonds.
The increased thermal agitation of the molecule
is sufficient to break most of the hydrogen bonds holding together the
3D tertiary protein structure, but NOT the
primary structure of the amino acid residues, which is dependant on the
strong covalent bonding of the polypeptide -NH-CO- linkages. The
denaturing of a protein can lead to its precipitation or coagulation
(cooking egg white). However in some cases on mild heating, the 3D
tertiary structure is 'unwound' and reforms on heating so the randomised
and stereospecific conformations are reversible. This means that the
primary 1D structure of amino acid residue sequence offers the
'blueprint' of a thermodynamically stable and specific 3D tertiary
conformation at lower temperatures, typically <50oC.
Alpha-helix conformation in protein-polypeptide structure
Enzyme tertiary structure
destroyed by e.g. heating which breaks disulphide linkages and
The full 3D structure of enzyme
intact and active site 'enabled'.
denaturing 'agents', which particularly affect the hydrogen bonding
of the tertiary structure e.g. hydrogen bonded alcohol competes with side-chain hydrogen bonding of
the proteins structure, hence its use as a disinfectant by denaturing
the enzymes in bacteria. Other tertiary structure denaturing 'agents'
include detergents and acids/alkalis and pH change are discussed
below in sections 3. ionic bonding, and
4. covalent bonding and reference back to this diagram will help follow the ideas.
One more example -
silk and wool - made of polypeptides, natural polyamides
Reminder of the condensation polymerisation process
brought about by 'polymerase' enzymes which
effect the elimination of water between the amino group (NH2)
and the carboxylic group (COOH) of two amino acids.
Silk and wool are examples of natural polyamides
Many active molecules and structures in
living organisms are polyamides.
Several of them are 'harvested' as
useful materials for use in fabrics for clothing.
Silk and wool are made of polyamides.
Silk contains a high proportion of
peptide links between serine molecules, an amino acid.
Serine has the formula HOCH2CH(NH2)COOH,
(A molecule with three functional
Wool has a high proportion of the
amino acid residue glycine H2NCH2COOH in its
Why silk and wool form strong fibres?
(see diagram above)
Natural wool and silk, and the
synthetic fibre Nylon have a β-sheet structure.
The protein polymer chains are
arranged in such a way that the fibre is like a stack of sheets of
corrugated cardboard held together by hydrogen bonds between the
δ- of the oxygen atom of the >C=O group and the δ+ hydrogen of the N-H
You can represent the hydrogen
and they are the strongest type of intermolecular bonding force
between adjacent molecules - in this case polypeptides.
Don't confuse this intermolecular
hydrogen bond with the strong intramolecular covalent bonding
of the peptide linkage
TOP OF PAGE
between polypeptide chains
This can occur
between ionisable side chains
e.g. a carboxylic group in one side chain can donate a proton to an
amino group in another side-chain so that ionisation occurs and the
formation of an ionic bond via the attraction of the positive and
negative ions. It is a strong bond and contributes significantly to the
maintenance of the 3D tertiary structure i.e.
However if the
pH is changed by making the enzyme's medium more acid or more
alkaline, then these ionic bonds can be disrupted i.e. the protein can be
'denatured' becoming less effective. The equations below illustrate the
behaviour of side-chains (and polypeptide chain terminal
functional groups) and the possible disruption of ionic
bonds or hydrogen bonding will affect the protein structure,
hence affect an enzymes function. In passing, they also illustrate
how proteins can act as buffering agents (buffers minimise
pH change if small amounts of acids/alkalis are added to a system).
the equations/structures only show a single residue and the chemical changes
of the side-chain e.g.
aspartic acid, 2-aminobutanedioic acid, residue Asp
which with alkali,
so increase in
pH (more alkaline) could
disrupt a hydrogen bond involving the HOOC group,
or with acid,
so decrease in
pH could disrupt
an important ionic bond.
covalent bonds connect one residue via the CO/NH link to the next amino acid
polypeptide link between two residues is NH-CO and
-COOH or -NH2 in the side chain.
glutamic acid, 2-aminopentanedioic acid,
so increase in
disrupt a hydrogen bond involving the HOOC group,
or with acid,
so decrease in
pH could disrupt
an important ionic bond.
lysine, 2,6-diaminohexanoic acid, residue Lys,
so decrease in
disrupt a hydrogen bond involving the H2N group.
in pH could disrupt
an important ionic bond.
This term should only be used in the context of ion solution of e.g.
KNO3(aq) or NH4NO3(aq) to complete
the circuit in an electrochemical cell.
Unfortunately, the term is also used in biochemistry to
describe the ionic bond above. Perhaps the term salt bridge bond
or an ionic bridge bond would be better? I've also come
across the term used to describe cations like Na+(aq)
loosely holding together proteins with a negative group in a side
chain but I can't
find any specific examples? Perhaps it is has been used to describe
the zinc ion, Zn2+(aq), holding six insulin
protein units together to form the quaternary hexamer. Here, a polar
group atom (lone pair donor) from each insulin molecule ,forms a
dative covalent bond with the zinc ion to form a complex ion,
bonding between polypeptide chains (usually disulphide linkage)
example is the S-S bond and occurs three times in the hormone
insulin, where two S-S bonds connect two polypeptide chains
together. The amino acid containing the original S-H bond is
cysteine and the sulphur-hydrogen groups of two neighbouring
cysteine residues can be oxidised to give the S-S bond.
cysteine, residue Cys, HS-CH2-CH(NH)-C=O
so in terms of
the two residues, Cys, the
disulphide bridge link is formed on oxidation ...
+ [O] ==> -CO-CH(NH-)-CH2-S-S-CH2-CH(NH-)-CO-
Covalent bonds connect one residue via the CO/NH link to the next amino acid
polypeptide link between two residues is NH-CO
SH (thiol) and -S-S (disulphide link)
via the side chain.
Cofactors and enzyme function
Although some enzymes can function
without additional chemical species, many enzymes (E) require one or
more extra components
known as co-factors (C) to be catalytically
active when the enzyme is bound to the substrate (S) to convert it into products (P). There are
different types of cofactors, non of them are proteins and most are either regenerated if chemically changed in the process, or chemically unchanged at the end of the reaction.
The terms cofactor and coenzyme seem to be loosely
interchangeable on the web and in textbooks.
Incidentally, C can also be an
inhibitor/deactivator as well as an
'promoter/activator/effector' for an enzyme and this duality in behaviour can be
regulate the chemistry in living organisms using cofactors.
(i) Coenzymes: These
cofactors are non-protein organic molecules which bind reversibly
to the enzyme, unlike prosthetic groups. They are often vitamin or
nucleotide derivatives and
function as carriers to transfer atoms or functional groups from the
enzyme to a substrate.
in principle: E + C
EC + S
EC + P
Metal ions such
as Zn2+, Mg2+, Mn2+,
Fe2+/3+, Cu+/2+, are often described as co-enzymes
themselves. These ions may be needed to provide an electrical charge
to hold a substrate (via dative covalent/ionic bonds) or
participate in an electron transfer process involving a change in
oxidation state (Cu or Fe). They can serve as (a) part of the principal
catalytic site, (b) a bridging group to bind enzyme and substrate
together covalently/ionically, or (c) an agent stabilizing the conformation of the enzyme
protein in its catalytically active form, again via covalent/ionic
bonds. Enzymes/proteins requiring metal
ions are sometimes called metalloenzymes/metalloproteins.
(ii) Prosthetic groups:
non-protein organic cofactor groups which are strongly bound
(effectively irreversibly) to the
in principle: E-C + S
E-C + P
Sometimes the prosthetic groups can
reversibly or irreversibly form complexes with a metal ions such
as those listed above. e.g. The enzyme catalase is very
effective in decomposing hydrogen peroxide to water and oxygen. The
active site partly consists of a cofactor combination of the
a prosthetic group (porphyrin) co-ordinated with a metal ion of
iron. Iron is a classic transition metal and the reaction involves
changes in ligand structure/co-ordination, oxidation state as well
as HO-OH bond breaking. Other catalysts promote this
decomposition without the enzyme BUT the enzyme-cofactor complex
is far more efficient. The uncatalysed reaction has an
activation energy (Ea) of about 80kJmol-1,
catalysts like iron salts/MnO2/Pt reduce it to about
40-50kJmol-1 but catalase reduces Ea to <10
kJmol-1 and it works remarkably fast at room
temperature Catalase and other peroxidases save our cells from the perils of a free and
unwelcome powerful oxidising agent! Remember a catalyst provides a
different reaction pathway that lowers the activation energy, helps
bond breaking and so speeds up the reactions considerably at room
temperature but anything we create artificially in the
laboratory ain't a patch so far on 'mother nature'!
TOP OF PAGE
of enzyme activity
GREATLY SIMPLIFIED DESCRIPTIONS, and do not cover all possibilities e.g.
many enzyme catalytic processes
involve two substrates but they only get the briefest of mention. A full
analysis and description requires up to 2nd year undergraduate level
knowledge, but I hope to ILLUSTRATE via EXAMPLES the use of the terms
irreversible, reversible, non-competitive, uncompetitive and allosteric
in an enzyme inhibition, any comments, as ever, would be appreciated.)
Enzymes inhibitors (I) in some way reduce, or completely stop, the
catalytic activity of an enzyme. Various scenarios are described
below and their descriptors may well overlap to describe a particular
inhibition situation completely. To appreciate the points below, its
advisable to revise the structure and function of
an enzyme. Many successful pharmaceutical products are enzyme
KEY: E = enzyme
(including any co-factor), S = substrate,
P = product(s), ES = enzyme-substrate complex, EP
= enzyme-product complex,
inhibitor bound to active centre or some other part of the enzyme,
= enzyme-inhibitor complex and EIS
= enzyme-inhibitor-substrate complex. The active site/centre is
the part of the enzyme where the catalyzed reaction takes place. A
non-active site (with respect to S==>P) site may allow binding of an inhibitor
or regulator molecule.
versus EI(active site) complex formation.
E + S
E + P
E + I
- The competitive and reversible binding of inhibitor
to the active site.
- A competitive
inhibitor is a
similar shaped molecule*
compared to the substrate. It binds reversibly to the enzymes active site-centre via
and so preventing the binding of the substrate molecule in
sequence (i). Therefore the substrate and inhibitor compete for the active
site of the enzyme i.e. sequence (i) versus reaction (ii). Increasing the concentration of a
substrate can increase its chances of binding with the active site and
decrease the effect of the inhibitor (Le Chatelier's equilibrium
principle). This is an example of reversible inhibition.
inhibitor can be described as
- Example 1: In
the aerobic tricarboxylic acid (citric acid/Krebs)
respiratory cycle, the enzyme succinate dehydrogenase
helps oxidise (by H loss)
the butanedioate ion (succinate)
into the trans-butenedioate ion (fumarate).
-2[H] ==> -OOC-CH=CH-COO-
- (its actually:
succinate + E-FAD <==> fumarate + E-FADH2
where FAD is a cofactor-coenzyme functioning as a hydrogen acceptor
or 'oxidiser' and getting reduced in the process)
- In the catalytic
process the butandioate ion binds to the active centre of E
via the two negative oxygens and succinate dehydrogenase is
inhibited by many ions which have a similar
stereochemical structure to
 propanedioate ion (malonate),
ion (oxalate, well know poison!), and
the pyrophosphate(V) ion.
- Example 2: ?
E + S
E + P
- Sequence (i) leads
to product formation.
- The formation of EI or EIS complex
instead of EP.
E + I
- In reaction (ii) the
inhibitor binds in reversible non-competitive inhibition, NOT competitive.
- Reaction (iii) is non-competitive
and uncompetitive reversible inhibition.
E + I
- Reaction (iv) is irreversible
the inhibitor strongly binds strongly to the active or non-active site
on the enzyme.
inhibition involves reactions (ii) and (iii) versus
sequence (i), where I
binds to a site (NOT the 'active site') on the enzyme to give EI
can also bind to ES to give ESI. There is NO competition between the substrate and
inhibitor for the active site of the enzyme. In other words the inhibitor does not
have to be stereochemically like the substrate molecule to bind to the
active site (no need to be the key for the lock!) but its
effect on the conformation* prohibits the transformation of ESI into products.
essentially the tertiary structure.
- Note (a) In two substrate
systems the binding of I and S1 to give ES1I
can prevent the binding of S2 to form ES1S2
needed to form the product,
- OR ES1
might be formed and I and S2 compete for the 2nd
active site to give ES1I (no product) or ES1S2
(can give product). I'm afraid descriptors can get quite mixed in
- Note (b): In an example
of so called
uncompetitive inhibition, ONLY involves
reaction (iii) versus sequence (i).
I can bind to the ES
complex (but NOT to the free enzyme on its own) to give an
enzyme-substrate-inhibitor complex that cannot breakdown to
form products. It seems that the binding of the
substrate changes the enzyme conformation to allow the
binding of the inhibitor. The inhibitor doesn't stop the substrate
binding BUT the conformation changes prevent the S => P
transformation in the ESI complex.
- In a two substrate
reaction, ESI formation can prevent the binding of the 2nd
substrate to produce ES1S2,
required to breakdown into E and P.
- The term allosteric inhibition applies to the situation where an
inhibitor binds to an enzyme somewhere other than the active
site (but another stereospecific or allosteric site),
in doing so, changes the 3D conformation
active site. This prevents either the substrate binding to the active
site and being converted
into product or allowing ESI to form products. So non-competitive
inhibition is a form of allosteric inhibition.
- Note - this
situation can work in a proactive way i.e. an activator
or modulator molecule (C), a
cofactor, rather than an inhibitor (I), can
bind with the enzyme changing the conformation of the active
site to enable an ECS complex to form that can breakdown to give
products. These activation/deactivation processes are very
important in regulating many reactions in living
- In irreversible
inhibition (iv) only, the inhibiting molecules or
ions bind, usually covalently (E-I), with the active site,
or to some other part of the enzyme structure and is not
easily removed. Therefore, the enzyme is permanently
changed/deactivated by completely 'blocking' the active site
OR changing the enzymes conformation so it cannot perform
its 'lock' function (the allosteric site effect). The
rate of the permanent inhibition reaction (iv)
will initially compete with the rates of sequence (i) but
progressively the enzyme system will cease to function. Therefore the total enzyme function is inhibited
and cannot be restored by adding excess substrate as in
- EXAMPLES to
ILLUSTRATE inhibition, mainly non-competitive and
- Example 1:
Heavy metal ions.
- Cations such as lead
(Pb2+), silver (Ag+) or mercury (Hg2+)
bind irreversibly with the main polypeptide chain and
permanently change the enzyme conformation.
catalyses the decomposition of hydrogen peroxide.
==> 2H2O(l) + O2(g)
- Redox analysis:
H only ox. state (+1), but for oxygen the changes are:
(-1) in H2O2 to (-2) in
H2O and (0) in O2.
- Catalase is a
heme enzyme involving an iron(III) ion co-ordinated in
the active centre. The mechanism is not completely
understood but is reported as involving iron(III) and
iron(IV) complexes in a redox catalytic cycle typical of
homogeneous transition metal catalysis. Addition of iron
salts to the enzyme-peroxide mixture can enhance the
rate of reaction (promotion) but addition of copper(II)
ion salts reduce the reaction rate (inhibition). Whether
the Cu2+ ions bind to the -NH- in the primary
polypeptide chain or a side chain COO-
group, I don't know?, but whatever, some part of the
protein structure will be acting as a ligand
coordinating with the Cu2+ ion to change the
conformation of the enzyme. This change in shape
renders the enzyme ineffective. It is non-competitive
inhibition because it doesn't bind with the active
site, but I'm not sure whether it is
reversible or not?
- Its also
possible that metal ions can replace the 'essential ion'
at the active site which themselves cannot perform the
essential electron transfers and oxidation state
- Zn2+, Ca2+, Hg2+,
can react irreversibly with the thiol group (-SH) in the
side-chain R group of polypeptides in a non-'active site' on
an enzyme, particularly those in the nervous system.
- Heavy metal ions can form insoluble
salts with proteins via anionic carboxylic groups in the side-chains (or
terminal) which would not only change their conformation but precipitate
them from cell fluids. In doing so they may well break ionic bridging
bonds (polypeptide-NH3+ -OOC-polypeptide)
important for maintaining secondary and tertiary structure i.e. the
enzyme protein is denatured.
- This reaction can be
used to treat heavy metal poisoning e.g. if a heavy metal
ion solution was accidentally ingested, you could be given
(hopefully rapidly!) a protein solution like milk or
egg-white to form the heavy metal ion-salt and then swallow
an emetic to make you vomit the offending material.
- Silver nitrate and
mercury salts are used as a disinfecting agent because they
inhibit/denature enzymes in bacteria.
- Cadmium (Cd2+)
and mercury (Hg2+) can replace Zn2+ in
the cofactor group in of alkaline phosphatase which
hydrolyses esters of phosphoric acid (not sure whether this
is a reversible competitive example?).
- Example 2: The
enzyme cytochrome c oxidase is important in electron
transfer (redox) systems of cell respiration. The enzyme
contains a co-factor (prosthetic complex ion group)
involving the oxidation state change of Cu(I) <==> Cu(II) +
- Its function is
inhibited by cyanide ion (CN-) which
acts a ligand and covalently binds irreversibly to the copper ion group
interfering with the function of the active site and
inhibiting the electron transfer via the Cu oxidation state
change. Hence it interferes with respiration and rather
fatally "My dear Watson!" If ingested and a suitable anti-dote is rapidly
taken, which traditionally in laboratories was (is?), I
gather, a horrible tasting iron solution which forms a
complex with the cyanide ion before reaching the
enzymes, you may survive!
- Example 3: Aspirin acting as a non-steroid anti-inflammatory agent
- Aspirin inhibits an
enzyme called cyclooxygenase-2 (also known as COX-2 or PHG2
synthase) which is partly responsible making prostaglandins
which stimulate the inflammatory responses of pain and fever
etc. The chemistry, in principle!, is quite simple.
2-ethanoylhydroxybenzoic acid (acetylsalicylic acid
or aspirin!) is hydrolysed in such a way that the ethanoyl
group (CH3CO) is transferred from the aspirin to
esterify the HO- (ol or hydroxy) group in the side-chain of
a serine amino acid residue (Ser).
+ HO-Ser-E ==> HOOCC6H4OH +
- The reaction is
irreversible and the change in conformation* of the enzyme
makes it inactive, so reducing the sequence of chemical
events that leads to feelings of pain etc., but don't
worry, your body eventually re-synthesises the necessary
enzymes to maintain your sensory warning systems!
*The change in
conformation could arise from disruption of a hydrogen
bond or the more bulky ethanoyl group 'pushing'
neighbouring groups of atoms further apart.
- Example 4: Certain
organic molecules used as nerve
poisons for warfare and insecticides.
organofluorophosphorus molecules like
diisopropylfluorophosphate, covalently bind with acetylcholinesterase and completely deactivate an enzyme
essential for the function of the nervous system, e.g. nerve
impulses are blocked and you lose body control. The now
banned nerve gas Sarin, binds with the -OH group
on a serine amino acid residue (Ser) on the active site, so deactivating
the enzyme, BUT it does not bind to 27 other Ser residue
sites on the enzyme! You die from respiratory failure in a
few minutes via fatally accurate stereochemistry, Sarin
is a biochemical 'sniper'!
- basically: Sarin-F + H-O-Ser-E ==> Sarin-O-Ser-E + HF
- Example 5:
- Chemicals from fungi
can irreversibly deactivate RNA polymerase and inhibit
crucial synthesis reactions in cell chemistry.
- Example 6:
Complex or chelating agents can bind reversibly to a metal
ion essential for catalytic activity.
- e.g. EDTA, a
polydentate ligand, can form a
complex with Mg2+ (or probably most other M2+
ion) and is a case of reversible non-competitive
+ [EDTA]4-(aq) +
[E](inactive) + [Mg-EDTA]2-(aq)
- The Mg2+
was itself part of a metalloprotein complex ion, but the
removal of the metal ion and change in enzyme ligand status, deactivates the
- The cyanide ion,
CN-, is a monodentate ligand and binds to the
iron(III) ion of the active site of
catalase (see also above) in competitive
- Example 7: Oxidising and reducing agents.
- Cells do not
generally like ingested 'foreign' strong oxidising or
reducing agents as they produce irreversible effects and
don't normally form a food
- One protein-enzyme
structure that is easily affected is the disulphide link
(-S-S-) or the thiol group (-SH) in the cysteine amino-acid
- Oxidation will form
a disulphide bond which would inevitably change the
conformation of an enzyme.
- 2 polypeptide-Cys-SH
+ [O] ==> polypeptide-Cys-S-S-Cys-polypeptide + H2O
- Or, a reducing agent
can break a necessary disulphide link needed to maintain the
secondary or tertiary structure of the enzyme protein.
- polypeptide-Cys-S-S-Cys-polypeptide + 2[H] ==> 2
The effects of
temperature increase and pH change and
protein denaturing effects on proteins are discussed elsewhere on the page
and a very
simplified discussion of enzyme
kinetics is on the advanced rates of reaction page.
TOP OF PAGE
simplified view of the structure and function of the protein haemoglobin
is NOT an enzyme, but does
consists of four polypeptide chains (the globin proteins) held
together as a tetramer (its quaternary structure) by intermolecular
forces. Each polypeptide protein chain has a heme/haem
cofactor group (which also occur in enzymes) which
consists of a 6 co-ordinated Fe2+ complex, so there are six
bonds in an 'octahedral arrangement, typical of a transition metal
complex. Five of the ligand bonds involve dative covalent bonding via lone
pairs from a nitrogen atom (the structure of the organic R group is
complex and omitted but involves lone pair donation from N or O). Two of the polypeptides are alpha-helixes and the
other two are beta-pleated sheets. When one oxygen molecule binds to one
of the four polypeptide chains, the shape is changed slightly to
facilitate the addition of three other oxygen molecules (a sort of
is a crude representation of the tetramer haemoglobin molecule
If present, and unfortunately, carbon monoxide is a more strongly
bonded ligand than O2, and will replace it. The CO will stay on
the haemoglobin molecule as carboxyhaemoglobin, inhibiting further
O2 co-ordination and hence cell respiration!
(i) If the Fe2+ is oxidised to Fe3+, the haemoglobin
molecule cannot acts as an oxygen carrier.
(ii) The haem/heme group is similar to the grouping on the photosynthesis
molecule chlorophyll, which has a magnesium ion, Mg2+, at the
centre of the 'complex' rather than an Fe2+ ion, and is
attached to a long hydrocarbon tail' rather than a polypeptide. Its
interesting that both molecules are to do with 'energy' chemistry, so
maybe we are seeing molecular evolution connections?
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