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Enzymes
Enzymes are remarkable molecules that determine all the chemical transformation that
make and break covalent bonds in cells. They may speed up reactions by a factor of a million
or more, using a favourable decrease in enthalpy to offset a decrease in entropy when a
substrate binds.
2007: Discuss how enzymes use a range of chemical tools to increase the rates of reactions.
Use lysozyme to illustrate your answer.
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Enzymes achieve extremely high rates of chemical reaction. They do this by
increasing the local concentration of substrate molecules, and by holding
appropriate atoms in the correct orientations. More importantly however, some of
the binding energy contributes directly to the catalysis. They provide an alternative
reactions pathway of lower activation energy, by passing substrate molecules
through a series of intermediate steps of altered geometry and electron distribution.
The activation energy is the free energy required to attain the most unstable
transition state.
Enzymes are unique in that they can use acid and base catalysis simultaneously – the
rigid framework of the protein binds the acidic and basic residues and prevents
them from combining with each other as they would do in solution.
Lysozyme was the first enzyme to have its structure worked out by x-ray
crystallography, and catalyses the cleavage of bacterial cell walls. These walls
contain peptidoglycan – a polymer of alternating NAG and NAM residues. NAM
moieties are linked to a short chain of amino acids (often D-amino acids to avoid
digestion by proteases) via a peptide bond. Amino acid chains are cross-linked to
each other to create a rigid layer of protein and carbohydrates. It is the
polysaccharide chain that is targeted by lysozyme.
The polysaccharide chain is broken by hydrolysis. However, in an aqueous solution
at room temperature, the energy of collisions with water molecules almost never
exceeds the activation energy, so hydrolysis occurs extremely slowly, if at all.
Geometric effects: The active site of lysozyme is a long groove that holds six linked
sugars at a time, designated A to F. Crucially, the fourth sugar (D) is forced into a
strained half-chair conformation (less stable, and so more reactive) rather than the
normal, stable chair form – this can occur because the favourable binding of the
other sugar rings offsets the strain.
As well as this physical distortion, there are two important amino acids strategically
placed in the active site. One is Glu-35, which is in a non-polar environment and so is
protonated – capable of acidic behaviour. The other is Asp-52, which is in a polar
environment and is negatively charged.
Asp-52, in its deprotonated form, can carry out nucleophilic attack on the C1 of the
D sugar. This displaces the oxygen of the glycosidic bond between D and E, and
forms a glycosyl-enzyme intermediate.
Glu-35 now donates a proton to the oxygen of C4 of E – this is acid catalysis.
To complete the hydrolysis, the now negatively charged Glu-35 activates a water
molecule which splits: the H+ re-protonates Glu-35, and the OH- reacts with C1 on D,
displacing Asp-52 to regenerate the enzyme. The reaction is complete.
The overall chemical reaction occurs millions of times faster than it would in the
absence of the enzyme.
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In other reactions involving two or more reactants, the active site also acts like a
template that brings the substrates together in the correct orientation for a reaction
to occur.
Other enzymes also may have a small molecule or metal atom tightly associated
with their active site that assists with their catalytic function. For example,
Carboxypeptidase (which cuts polypeptide chains) carries a tightly bound zinc ion in
its active site. The zinc ion forms a transient bond with one of the substrate atoms,
thereby assisting the hydrolysis reaction. Similarly, small organic molecules
(coenzymes) can assist reactions. Biotin is found in enzymes that transfer a COOgroup from one molecule to another. Biotin forms a transient covalent bond with
the –COO- group, being better suited to this function than any of the amino acids
used to make proteins. Hence other small cofactors are used to expand the (already
huge) range of possible chemical reactions that enzymes can catalyse.
Discuss the ways in which enzymes can be controlled.
A living cell contains thousands of different enzymes, operating at the same time in
the same small volume of the cytosol. They interact to generate a web of pathways
– this maze of reactions is so complex that elaborate controls are required to
regulate when and how rapidly each reaction occurs.
A very common and simple method: Reversible inhibition. If the inhibitor closely
resembles the substrate, it is a competitive inhibitor and can bind to the enzyme,
preventing the substrate from doing so. This does not change Vmax but Km is
increased. E.g. methotrexate mimics folic acid and competes with the cosubstrate
for thymine synthesis, tetrahydrofolate. It is used as a chemotherapy drug. AZT is a
HIV drug that once converted into AZT(TP) behaves as a chain terminator in cDNA
production by HIV reverse transcriptase by competing with deoxyATP.
Irreversible inhibition: These react with an enzyme to form a covalent adduct, and
usually target reactive amino acid sidechains in the active sites of enzymes. The
nerve agent sarin reacts with the active site serine in acetylcholinesterase, leading to
a build up of ACh in the synaptic cleft, so action potentials are constantly generated
and the inability to control breathing results in asphyxiation.
Suicide inhibitors resemble the substrate, but are chemically unreactive. The
enzyme forms a covalent bond with the inhibitor but the reaction is unable to
proceed further, and the enzyme is left with a covalently bound inhibitor in its active
site. Penicillin reacts with a serine in the active site of glycopeptide transferase,
preventing peptide bond formation in peptidoglycan cell wall synthesis.
Reversible covalent modification: The most common form is reversible
phosphorylation of the side chains of specific serine, threonine or tyrosine residues
in the target enzyme. Phosphorylation is itself catalysed by specific kinases, and
phosphate groups are removed by phosphatases.
Phosphorylation can result in a major conformational change because phosphates
have two negative charges, so for example can attract a cluster of positively charged
side chains, and this can affect the binding of ligands elsewhere on the enzyme,
dramatically changing the proteins activity. An attached phosphate can also form
part of a structure that other proteins recognise.
Phosphorylation is vital for regulating all sorts of processes from metabolism to
development. In the cell cycle, specific phosphorylation events (by CAK and Wee1)
and dephosphorylation (by Cdc25) activeate M-Cdk, which goes on to phosphorylate
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and trigger various steps in the initiation of mitosis. For example phosphorylation of
the anaphase promoting complex allows its activation by cdc20 binding, resulting in
a cascade of events that initiate the separation of sister chromatids.
Long term control can be achieved with by genetic control. Excess tryptophan
represses the production of all the enzymes in the tryptophan biosynthesis pathway,
using negative feedback to prevent the cell wasting energy.
Allosteric enzymes are very important and do not obey Michaelis-Menten kinetics.
These have several subunits that bind cooperatively to the substrate. The affinity of
the enzyme for the substrate depends on whether there is substrate already bound.
At low substrate concentrations, the affinity is low. However when one substrate
molecule binds, others can now bind more easily. A sigmoidal Michaelis-Menten
plot is produced. Allosteric activators shift the sigmoidal curve to the left, while
allosteric activators shift it to the right. This is possible because allosteric enzymes
have several subunits and more than one active site.
PFK is involved in glycolysis, and converts fructose-1-phosphate to fructose-1,6bisphosphate. It has four active sites and several allosteric effectors. When energy is
needed, AMP concentrations are high, and this activates glycolysis by shifting the
curve to the left. Conversely, when ATP levels are high and energy is not in high
demand, ATP inhibits PFK and glycolysis by shifting the curve to the right.
One of the best-studied examples is aspartate transcarbamoylase. This catalyses the
important reaction between carbamoyl phosphate and aspartate that begins
pyrimidine synthesis. One of the final products of this pathway is CTP. Binding of the
substrates (carbamoyl phostphate and aspartate) drive a large conformational
change, switching the enzyme into its active R form, from which regulatory CTP
molecules dissociate. By contrast, binding of CTP converts the enzyme into its
inactive T conformation. This enables the enzyme to be turned on suddenly as
substrates accumulate, or be turned off when CTP accumulates. Here the allosteric
enzymes undergoes a concerted, all-or-none transition rather than a sequential one.
Understanding how enzymes work not only enables us to figure out how extremely
complex organisms such as ourselves work and have evolved, but understanding
their regulation and control is extremely important in medicine, since enzyme
inhibitors make effective drugs against many devastating diseases.