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Lecture 8

BIOL 200 Lecture Notes - Lecture 8: Rate Equation, Combinatorial Chemistry, Cgmp-Specific Phosphodiesterase Type 5

Biology (Sci)
Course Code
BIOL 200
Mathieu Roy

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As shown in the figure to the left, irreversible inhibitors form a reversible non-covalent complex
with the enzyme (EI or ESI) and this then reacts to produce the covalently modified "dead-end
complex" EI*. The rate at which EI* is formed is called the inactivation rate or kinact. Since
formation of EI may compete with ES, binding of irreversible inhibitors can be prevented by
competition either with substrate or with a second, reversible inhibitor. This protection effect is
good evidence of a specific reaction of the irreversible inhibitor with the active site.The binding
and inactivation steps of this reaction are investigated by incubating the enzyme with inhibitor
and assaying the amount of activity remaining over time. The activity will be decrease in a time-
dependent manner, usually following exponential decay. Fitting these data to a rate
equation gives the rate of inactivation at this concentration of inhibitor. This is done at several
different concentrations of inhibitor. If a reversible EI complex is involved the inactivation rate
will be saturable and fitting this curve will give kinact and Ki. Another method that is widely used
in these analyses is mass spectrometry. Here, accurate measurement of the mass of the
unmodified native enzyme and the inactivated enzyme gives the increase in mass caused by
reaction with the inhibitor and shows the stoichiometry of the reaction. This is usually done
using a MALDI-TOF mass spectrometer. In a complementary technique, peptide mass
fingerprinting involves digestion of the native and modified protein with a proteasesuch
as trypsin. This will produce a set of peptides that can be analysed using a mass spectrometer.
The peptide that changes in mass after reaction with the inhibitor will be the one that contains the
site of modification.
Not all irreversible inhibitors form covalent adducts with their enzyme targets. Some reversible
inhibitors bind so tightly to their target enzyme that they are essentially irreversible. These tight-
binding inhibitors may show kinetics similar to covalent irreversible inhibitors. In these cases,
some of these inhibitors rapidly bind to the enzyme in a low-affinity EI complex and this then
undergoes a slower rearrangement to a very tightly bound EI* complex (see figure above). This
kinetic behaviour is called slow-binding. This slow rearrangement after binding often involves
a conformational change as the enzyme "clamps down" around the inhibitor molecule. Examples
of slow-binding inhibitors include some important drugs, such methotrexate, allopurinol, and the
activated form of acyclovir.
Diisopropylfluorophosphate (DFP) is shown as an example of an irreversible protease inhibitor
in the figure above right. The enzyme hydrolyses the phosphorusfluorine bond, but the
phosphate residue remains bound to the serine in the active site, deactivating it. Similarly, DFP
also reacts with the active site of acetylcholine esterasein the synapses of neurons, and
consequently is a potent neurotoxin, with a lethal dose of less than 100 mg. Suicide inhibition is
an unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive
form in its active site. An example is the inhibitor of polyamine biosynthesis, α-
difluoromethylornithine or DFMO, which is an analogue of the amino acidornithine, and is used
to treat African trypanosomiasis (sleeping sickness). Ornithine decarboxylase can catalyse the
decarboxylation of DFMO instead of ornithine, as shown above. However, this decarboxylation
reaction is followed by the elimination of a fluorine atom, which converts this catalytic
intermediate into a conjugated imine, a highly electrophilic species. This reactive form of DFMO
then reacts with either a cysteine or lysine residue in the active site to irreversibly inactivate the
enzyme. Since irreversible inhibition often involves the initial formation of a non-covalent EI
complex, it is sometimes possible for an inhibitor to bind to an enzyme in more than one way.
For example, in the figure showing trypanothione reductase from the human protozoan
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