Tutorial 4 Solutions

1) Lineweaver-Burke plots are shown below when there is no inhibitor present in an

enzymatic reaction with a substrate.
a) Draw on the graph the new plot when an inhibitor is added in each case. What do
the x and y intercepts represent? State what happens to the Km and Vmax of the
enzyme-substrate complex in each case.

Competitive inhibition

Non-competitive inhibtion

Uncompetitive inhibition
Competitive inhibition: Vmax unaffected, Km increases. ‘Apparent’ Km increases
because the inhibitor competes with the substrate, so the enzyme’s affinity for the
substrate appears to be lowered, particularly at low [S]. At low [S], the inhibitor-
enzyme complex forms, and stops substrate from binding. The Vmax stays the
same, because at high concentration, the substrate will outcompete the inhibitor (the
inhibitor concentration is fixed!). At low substrate concentrations, the inhibitor
influences the reaction rate, hence the change in rate at lower [S].

Non-competitive inhibition: Inhibitor binds at an allosteric site. Substrate still binds to

enzyme, but catalytic reaction cannot occur anymore. Presence of inhibitor doesn’t
affect the affinity of the substrate, but it stops the catalytic reaction from happening.
Thus Km stays the same, but Vmax decreases.

Uncompetitive inhibitor: Inhibitor binds only to the enzyme-substrate complex.

Inhibition depends on both inhibitor concentration and substrate concentration. Vmax
decreases, as higher substrate concentration increases inhibition rate. Km
decreases, the apparent affinity of the substrate appears higher. This is because the
more enzyme substrate complex forms, the more enzyme-substrate-inhibitor
complex forms.

b) You are investigating the inhibition of a kinase protein that binds to ATP. You
incubate the kinase with the inhibitor, and increasing concentrations of ATP, but find
that the Vmax stays the same in each instance. What type of inhibition is likely taking
place?
The Vmax stays the same when a higher conc of ATP substrate is added – therefore,
this must be competitive inhibition.

c) What is Ki, what does it mean, and what other equilibrium constant is it
comparable to?
Ki is the inhibition constant, being the concentration required for 50% occupancy of
the inhibitor target. It is comparable to Kd, but Kd is typically used to describe a ligand
rather than an inhibitor.

d) You have three inhibitors each with different Ki values of 250 nM, 2 µM, and 200
µM. Rank these inhibitors by their Ki values, from tightest binding to weakest.
250 nM strongest
2 uM middle
200 uM weakest

2) What are the benefits and limitations of covalent inhibitors with respect to non-
covalent inhibitors.

Benefits Limitations
Increased efficiency Selectivity
Independent of [S] Off target effects
Long lasting effects Toxicity
Effective at lower doses

3) Your covalent inhibitor undergoes a reaction with a cysteine residue. What is the
side chain functional group of Cys?

Thiol HS

HN

4) Given that your inhibitor has a conjugated ketone, draw the mechanism of
addition.
O

HS E
R
 O
HS E
R

S
R

H E

R S E

5) Penicillins were the first antibiotics described, having since saved many lives.
What function do penicillins inhibit? Why are penicillins so reactive? And what
enzyme has given bacteria resistance to penicillins?
• Cell wall biosynthesis – specifically crosslinking between chains
• The beta-lactam results in ring strain and a lack of resonance across the
amide
• Beta-lactamases – undergo same initial reaction as D-Ala-D-Ala
carboxypeptidase, but then are able to hydrolyse the resulting ester
6) Why is the below inhibitor (5-fluoro-2’-deoxycytidine) capable of inhibiting the
reaction shown?
During the reaction there is loss of a
proton. The inhibitor has a fluorine in
place of the hydrogen. Fluorine is
very electronegative. Would require
fluorine to leave as a cation.
Therefore the reaction becomes stuck
and the enzyme is covalently bound
to the inhibitor and is unable to bind
any substrate and catalyse any
reactions.