Advanced A Level Organic Chemistry: Stereochemistry - polypeptide and protein structure

Scroll down and take time to study the content and/or follow links or [Use the website search box]

Doc Brown's Advanced A Level Organic Chemistry Revision Notes - Help in Revising Advanced Organic Chemistry

PART 14 ORGANIC ISOMERISM and Stereochemistry Revision Notes

Part 14.4 Notes on protein structure, enzyme structure and function and metalloenzymes

Case studies of structure, formation, properties and consequences

email doc brown - comments - query?

All my advanced A level organic chemistry notes

All my advanced A level isomerism and stereochemistry notes

Use your mobile phone or ipad etc. in 'landscape' mode

This is a BIG website, you need to take time to explore it [SEARCH BOX]

14.4 Stereoisomerism, aspects of stereochemistry contd.

this page 14.4:  2b.6 sub-index: of notes on protein/enzyme structure and function

introduction * enzyme function * protein structure - denaturing * structure of silk and wool

enzyme co-factors/co-enzymes * enzyme inhibition * haemoglobin

other pages: Introduction to E/Z (geometric) and R/S (optical) isomerism :

other pages: 2b.1 amino acids * 2b.2 alanine synthesis * 2b.3 lactic acid synthesis  2b.4 Thalidomide

2b.5 nucleophilic substitution of halogenoalkanes* 14.6 isotactic/atactic/syndiotactic poly(propene)

combinational chemistry and autosynthesis * protein hydrolysis and analysis

14.4 Case studies - Extra notes on protein/enzyme structure and function

Forward 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 very sick!

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 substrates (reactants).

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 below.

See also: GCSE notes enzyme rates of reaction and uses and GCE-AS-A2 notes on enzyme kinetics

The importance 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 sequences.

The 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

2-hydroxybutanedioic 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 and sub-index

Protein Structure

The importance of proteins

Proteins are extremely important molecules in living systems and very little biochemistry happens without them being involved.

They are 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

Fibrous 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.

Transporters of other molecules through membranes.

Regulatory roles are also performed by globular proteins rather than fibrous proteins.

(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.

Membrane receptor proteins relay signals between the cell's internal and external environments.

Transport proteins move molecules and ions across the membrane.

Membrane enzymes 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 described.

Primary structure of proteins:

The order of the amino acids in the polypeptide chain (the sequence of residues). Polypeptide synthesis-formation is illustrated in a later section. The primary 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.

Note the 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 link.

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).


diagram is to show you how to read the sequence of amino acid residues in a polypeptide and identify the original amino acid molecular structure from the protein structure

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  or HOOC-CH2-CH(NH2)-COOH


Secondary structure of proteins:

The coiling or folding of the polypeptide chain into an a-helix or the formation of a beta-pleated sheet of protein is stabilised by hydrogen bonding (see diagrams below).

Alpha-helix form of proteins

secondary structure of proteins alpha helix form of polypeptides held together by hydrogen bonding doc brown's advanced level organic chemistry revision notes

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 context:

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 structure.

Beta-pleated sheet form of proteins

HYDRPOGEN BONDING and skeletal formula of polypeptide-protein beta sheets (c) doc b

In these protein structures the polypeptide chains are mostly fully extended and held by intermolecular hydrogen bonds to other polypeptide chains (see structure of silk and wool). Intermolecular means 'between molecules'

i.e. in this context: polypeptide1>C=Oδ-llllδ+H-N<polypeptide2

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.


Tertiary structure of proteins:

tertiary structure of proteins 3D form of polypeptide held together by hydrogen bonding covalent S-S and ionic bonds COO- +NH3doc brown's advanced level organic chemistry revision notes

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 3D structure.

The tertiary 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' groups (see examples below).

Enzymes, for example, might incorporate both alpha helix and beta sheet sections of polypeptide structures.


Quaternary structure of proteins:

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 hexamer (insulin)6.

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 haemoglobin holds 4 components together.


Four types of interaction are important in holding together the secondary and tertiary structure.

(i) Instantaneous 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

(iii) Ionic bonding - ionisable side chain groups e.g. between  -COO-  and   +NH3-  groups.

(iv) Covalent bonding e.g. disulfide bridges -S-S-.

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. See also a discussion of enzyme kinetics - rates of reaction

TOP OF PAGE and sub-index

Secondary or tertiary structure of proteins and denaturing (and enzyme inhibition)

Examples of secondary or tertiary structures are described below and references to denaturing 

1. Instantaneous/transient dipole-induced dipole forces between polypeptide chains

Non-polar Van der Waals forces, hydrophobic interactions in the presence of water.

These forces arise from the random behaviour of the electron fields in atoms producing minute transient dipoles δ+moleculeδ- 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 substrate molecules.

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.

(a) leucine (c) doc b leucine, 2-amino-4-methylpentanoic acid,

  residue Leu, (CH3)2CHCH2CH(NH-)CO-

(b) isoleucine (c) doc b isoleucine, 2-amino-3-methylpentanoic acid,

residue Ile, CH3CH2CH(CH3)CH(NH-)CO-

(c) phenylalanine (c) doc b phenylalanine, 2-amino3-phenylpropanoic acid,

residue Phe, C6H5CH2CH(NH-)CO-

and remember 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.

These weak instantaneous 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 and sub-index

2. Polar bonds and hydrogen bonding between polypeptide chains

Polar permanent dipole-permanent dipole intermolecular forces, hydrophilic interactions in the presence of water.

HYDRPOGEN BONDING in polypeptides-proteins (c) doc bPermanent 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 δ+H-Nδ- 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 forces.

[1] Hydrogen bonding between an alcohol group in the side chain of an amino acid residue and water surrounding a protein molecule.

[2] Hydrogen bonding between an alcohol group in the side chain of an amino acid residue and an amine group in a side-chain.

[3] Hydrogen bonding between a carbonyl group and water with a protein in an aqueous media.

[4] 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 denaturing diagram) OR between two separate polypeptide chains held together e.g. beta-sheets of protein material, structure shown schematically below.

HYDROGEN BONDING and structural formula of polypeptides-proteins beta sheets (c) doc b

Polypeptide 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.

HYDRPOGEN BONDING and skeletal formula of polypeptide-protein beta sheets (c) doc b

A 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 bonds).

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.

These 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 hydrogen bonding between neighbouring highly polar groups. For example, δ-O-Hδ+, δ-NH2δ+ or δ+C=Oδ- groups in the side chains of serine, threonine, asparagine and glutamine.

look for the possibility of  δ-O-Hδ+||||δ-O=Cδ+, δ-O-Hδ+||||δ+H-Nδ-, δ-N-Hδ+||||δ-O=Cδ+, δ-O-Hδ+||||etc.

(a) serine (c) doc b serine, 2-amino-3-hydroxypropanoic acid

residue Ser, -Oδ-||||Hδ+-Oδ-CH2CH(NH-)CO-

the purple |||| shows just one exemplar hydrogen bond, Hδ+-Oδ-|||Hδ+-Oδ-, and 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 residue, the polypeptide linkage between two residues is NH-CO.

(b) threonine (c) doc b threonine, 2-amino-3-hydroxybutanoic acid

residue Thr, CH3CH(OH)CH(NH-)CO-, with an H-O on a side chain

(c) asparagine (c) doc b asparagine,

residue Asn, H2N-C(=O)CH2CH(NH-)CO-, with a H2N or C=O on a side chain

(d) glutamine (c) doc bglutamine,

residue Gln, H2N-C(=O)CH2CH2CH(NH-)CO-, with a H2N or C=O on a side chain

(e) aspartic acid (c) doc b aspartic acid, 2-aminobutanedioic acid,

residue Asp, HOOCCH2CH(NH-)CO-, with an OH or C=O on a side chain

(f) lysine (c) doc b lysine, 2,6-diaminohexanoic acid,

residue Lys H2NCH2CH2CH2CH2CH(NH-)CO-, with a H2N on a side chain


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 (usually >45oC).

Again, 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 primary structure.

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 and tertiary 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.

(i) alpha-helix conformation in protein-polypeptide structure, (ii) enzyme tertiary structure destroyed by e.g. heating which breaks disulphide linkages and hydrogen bonds, (iii) full 3D structure of enzyme intact and active site 'enabled'. (c) doc b

  1. Alpha-helix conformation in protein-polypeptide structure

  2. Enzyme tertiary structure destroyed by e.g. heating which breaks disulphide linkages and hydrogen bonds

  3. The full 3D structure of enzyme intact and active site 'enabled'.

Other 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.

formation of peptide linkage in polypeptide proteins from amino acids by elimination of water

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, 2-amino-3-hydroxypropanoic acid.

(A molecule with three functional groups)

polypeptide protein molecule of serine in silk peptide linkage molecular structure of 2-amino-3-hydroxypropanoic acid amino acid structural formula

Wool has a high proportion of the amino acid residue glycine H2NCH2COOH in its molecules.

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 group.

You can represent the hydrogen bonds as >N-Hδ+llllδ-O=C< 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 >NH-CO-.

TOP OF PAGE and sub-index

3. Ionic bonding 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.

R'-NH2 + HOOC-R  R'-NH3+ + -OOC-R

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 acid-base 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). Note: the equations/structures only show a single residue and the chemical changes of the side-chain e.g. 

 (a) aspartic acid (c) doc b aspartic acid, 2-aminobutanedioic acid, residue Asp

which with alkali, HOOCCH2CH(NH-)CO- + OH- -OOCCH2CH(NH-)CO- + H2O

so increase in pH (more alkaline) could disrupt a hydrogen bond involving the HOOC group,

or with acid, -OOCCH2CH(NH-)CO- + H+ HOOCCH2CH(NH-)CO-

so decrease in pH could disrupt an important ionic bond.

The covalent bonds connect one residue via the CO/NH link to the next amino acid residue, the polypeptide link between two residues is NH-CO and the -COOH or -NH2 in the side chain.

(b) glutamic acid (c) doc b glutamic acid, 2-aminopentanedioic acid, residue Glu,

with alkali, HOOCCH2CH2CH(NH-)CO- + OH- -OOCCH2CH2CH(NH-)CO- + H2O

so increase in pH could disrupt a hydrogen bond involving the HOOC group,

or with acid, -OOCCH2CH2CH(NH-)CO- + H+ HOOCCH2CH2CH(NH-)CO-

so decrease in pH could disrupt an important ionic bond.

(c) lysine (c) doc b lysine, 2,6-diaminohexanoic acid, residue Lys

with acid, H2NCH2CH2CH2CH2CH(NH-)CO- + H+ +H3NCH2CH2CH2CH2CH(NH-)CO-

so decrease in pH could disrupt a hydrogen bond involving the H2N group.

with alkali +H3NCH2CH2CH2CH2CH(NH-)CO- + OH- H2NCH2CH2CH2CH2CH(NH-)CO- + H2O

so increase in pH could disrupt an important ionic bond.

Salt bridge: 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, [Zn(:insulin)6]2+(aq).

4. Covalent bonding between polypeptide chains (usually disulphide linkage)

One common 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 (c) doc b 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 ...

2 HS-CH2-CH(NH-)-CO- + [O] ==>  -CO-CH(NH-)-CH2-S-S-CH2-CH(NH-)-CO- + H2O

Covalent bonds connect one residue via the CO/NH link to the next amino acid residue, the polypeptide link between two residues is NH-CO and 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 used to 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 reversible EC + S reversible ECS reversible 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:

These non-protein organic cofactor groups which are strongly bound (effectively irreversibly) to the protein.

in principle: E-C + S reversible E-CS reversible 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 and sub-index

The inhibition of enzyme activity

(These are 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 inhibitors.

KEY: E = enzyme (including any co-factor), S = substrate, P = product(s), ES = enzyme-substrate complex, EP = enzyme-product complex, I = inhibitor bound to active centre or some other part of the enzyme, EI = 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.

  • Competitive inhibition: ES versus EI(active site) complex formation.

    • (i) E + S ES EP E + P
      • Product formed.
    • (ii) E + I EI(active site)
      • 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 (ii), 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. *A competitive inhibitor can be described as 'substrate analogue'.
    • Example 1: In the aerobic tricarboxylic acid (citric acid/Krebs) respiratory cycle, the enzyme succinate dehydrogenase helps oxidise (by H loss) [1] the  butanedioate ion (succinate) into the trans-butenedioate ion (fumarate).
      • -OOC-CH2-CH2-COO- -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 it e.g.
      • [2] propanedioate ion (malonate),  [3] ethanedioate ion (oxalate, well know poison!), and [4] the pyrophosphate(V) ion.
      • [1] the butanedioate ion (succinate), [2] propanedioate ion (malonate),  [3] ethanedioate ion (oxalate), and [4] the pyrophosphate(V) ion. (c) doc b
    • Example 2: ?
      • ?
  • Non-competitive/uncompetitive/irreversible inhibition:

    • (i) E + S reversible ES reversible EP reversible E + P
      • Sequence (i) leads to product formation.
    • The formation of EI or EIS complex instead of EP.
    • (ii) E + I reversible EI(non-active site)
      • In reaction (ii) the inhibitor binds in reversible non-competitive inhibition, NOT competitive.
    • (iii) ES + I ESI(non-active site)
      • Reaction (iii) is non-competitive and uncompetitive reversible inhibition.
    • (iv) E + I E-I(active/non-active site)
      • Reaction (iv) is irreversible inhibition, the inhibitor strongly binds strongly to the active or non-active site on the enzyme.
    • Noncompetitive 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 AND 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 inhibition.
      • 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 ESI 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), and in doing so, changes the 3D conformation around the 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 organisms.
    • 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 competitive inhibition.
    • EXAMPLES to ILLUSTRATE inhibition, mainly non-competitive and irreversible.
    • Example 1: Heavy metal ions.
      • Cations such as lead (Pb2+), silver (Ag+) or mercury (Hg2+) can strongly bind irreversibly with the main polypeptide chain and permanently change the enzyme conformation.
        • Catalase catalyses the decomposition of hydrogen peroxide.
        • 2H2O2(aq) ==> 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 changes.
      • Zn2+, Ca2+, Hg2+, Pb2+ 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) + e-.
      • 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).
      • HOOCC6H4OCOCH3 + HO-Ser-E ==> HOOCC6H4OH + CH3COO-Ser-E
      • 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.
      • Some 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: Fungal toxins.
      • 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 inhibition.
      • [E-Mg]2+(active) + [EDTA]4-(aq) + reversible [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 enzyme.
      • 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 inhibition.
    • 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 constituent.
      • One protein-enzyme structure that is easily affected is the disulphide link (-S-S-) or the thiol group (-SH) in the cysteine amino-acid residue (Cys).
      • 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 polypeptide-Cys-SH

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 and sub-index

A simplified view of the structure and function of the protein haemoglobin

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 molecular co-operation).

[ alpha/beta-polypeptide-[Fe2+(:NR5)O2] ]4 is a crude representation of the tetramer haemoglobin molecule

toxic 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!

Note: (i) If the Fe2+ is oxidised to Fe3+, the haemoglobin molecule cannot acts as an oxygen carrier.

Note (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?  

KS3 BIOLOGY QUIZZES ~US grades 6-8 KS3 CHEMISTRY QUIZZES ~US grades 6-8 KS3 PHYSICS QUIZZES ~US grades 6-8 HOMEPAGE of Doc Brown's Science Website EMAIL Doc Brown's Science Website
GCSE 9-1 BIOLOGY NOTES GCSE 9-1 CHEMISTRY NOTES and QUIZZES GCSE 9-1 PHYSICS NOTES GCSE 9-1 SCIENCES syllabus-specification help links for biology chemistry physics courses IGCSE & O Level SCIENCES syllabus-specification help links for biology chemistry physics courses
Advanced A/AS Level ORGANIC Chemistry Revision Notes US K12 ~grades 11-12 Advanced A/AS Level INORGANIC Chemistry Revision Notes US K12 ~grades 11-12 Advanced A/AS Level PHYSICAL-THEORETICAL Chemistry Revision Notes US K12 ~grades 11-12 Advanced A/AS Level CHEMISTRY syllabus-specificatio HELP LINKS of my site Doc Brown's Travel Pictures
Website content Dr Phil Brown 2000+. All copyrights reserved on revision notes, images, quizzes, worksheets etc. Copying of website material is NOT permitted. Exam revision summaries & references to science course specifications are unofficial.

 Doc Brown's Chemistry 


TOP OF PAGE and sub-index