TECHNOLOGICAL INSTITUTE OF THE PHILIPPINES

363 P. CASAL ST. QUIAPO, MANILA

BIOCHEMICAL ENGINEERING
HOMEWORK
ENZYME INHIBITORS, LINEWEAVER-BURK PLOT, EADIE-HOFSTEE PLOT, AND
HANES-WOOLF PLOT

MAURICIO, KRIZZIA ANNE C.

ENGR. FRANCIS SY

AUGUST 17, 2018

ENZYME INHIBITORS
Enzyme inhibitors are molecules that interact in some way with the enzyme to prevent it from working
in the normal manner. It disrupts the normal reaction pathway between an enzyme and a substrate (prevents
the formation of an enzyme-substrate complex and hence prevent the formation of product). There are a
variety of types of inhibitors including: Competitive, Non-competitive, Uncompetitive, Reversible, and
Irreversible Inhibitors.

Normal Enzyme Reaction

o In a normal reaction, a substrate binds to an enzyme (via the active site) to form an enzyme-substrate
complex.
o The shape and properties of the substrate and active site are complementary, resulting in enzyme-
substrate specificity.
o When binding occurs, the active site undergoes a conformational change to optimally interact with
the substrate (induced fit).
o This conformational change destabilizes chemical bonds within the substrate, lowering the activation
energy.
o As a consequence of enzyme interaction, the substrate is converted into product at an accelerated
rate.

Competitive Inhibition
o Competitive inhibition involves a molecule, other than the substrate, binding to the enzyme’s active
site.
o The molecule (inhibitor) is structurally and chemically similar to the substrate (hence able to bind to
the active site).
o The competitive inhibitor blocks the active site and thus prevents substrate binding.
 o As the inhibitor is in competition with the substrate, its effects can be reduced by increasing substrate
concentration.

Non-competitive Inhibition
o Non-competitive inhibition involves a molecule binding to a site other than the active site (an allosteric
site).
o The binding of the inhibitor to the allosteric site causes a conformational change to the enzyme’s
active site.
 o As a result of this change, the active site and substrate no longer share specificity, meaning the
substrate cannot bind.
o As the inhibitor is not in direct competition with the substrate, increasing substrate levels cannot
mitigate the inhibitor’s effect.

Substrate and inhibitor react with different active site of the enzyme.
Deactivating complex is formed by two reversible reaction steps.
For PURE non-competitive inhibition: KAI = KCI

For MIXEDnon-competitive inhibition: KAI ≠ KC

Uncompetitive Inhibition
It is also known as anti-competitive inhibition, takes place when an enzyme inhibitor binds only to the
complex formed between the enzyme and the substrate (the E-S complex). Uncompetitive inhibition typically
occurs in reactions with two or more substrates or products.

Reversible Inhibition
A reversible inhibitor is one that, once removed, allows the enzyme it was inhibiting to begin working
again. It has no permanent effects on the enzyme - it does not change the shape of the active site, for
example. Reversible Inhibition may be Competitive, Non-Competitive or Uncompetitive.

Irreversible Inhibition
An irreversible inhibitor will bind to an enzyme so that no other enzyme-substrate complexes can
form. It will bind to the enzyme using a covalent bond at the active site which therefore makes the enzyme
denatured. An example of an irreversible inhibitor is di-isopropyl fluoro-phosphate which is present in nerve
gas. It binds to the enzyme and stops nerve impulses being transmitted. An example of where we use
irreversible inhibitors in medicine is penicillin. Penicillin works by inhibiting the activity of the enzyme
responsible for the creation of the bacterial cell wall. This means that water can enter the bacterial cell,
causing it to swell, burst and die termed lysis.
 LINE-WEAVER-BURK PLOT

The Lineweaver–Burk plot was widely used to determine important terms in enzyme kinetics, such
as Km and Vmax;, before the wide availability of powerful computers and non-linear regression software. The
y-intercept of such a graph is equivalent to the inverse of Vmax; the x-intercept of the graph represents
−1/Km. It also gives a quick, visual impression of the different forms of enzyme inhibition.
The Lineweaver–Burk plot is classically used in older texts, but is prone to error, as the y-axis takes
the reciprocal of the rate of reaction – in turn increasing any small errors in measurement. Also, most points
on the plot are found far to the right of the y-axis (due to limiting solubility not allowing for large values of [S]
and hence no small values for 1/[S]), calling for a large extrapolation back to obtain xand y-intercepts.
Line-Weaver-Burk Plot commonly-used plot in examining enzyme kinetics, in with the inverse of the
reaction rate, 1/r, is plotted against the inverse of the substrate concentration 1/[S].
 EADIE-HOFSTEE PLOT

The Eadie–Hofstee plot is a graphical representation of enzyme kinetics in which reaction rate is
plotted as a function of the ratio between rate and substrate concentration and can be derived from the
Michaelis–Menten equation by inverting and multiplying with Vmax.
One drawback from the Eadie–Hofstee approach is that neither ordinate nor abscissa represent
independent variables: both are dependent on reaction rate. Thus any experimental error will be present in
both axes. Also, experimental error or uncertainty will propagate unevenly and become larger over the
abscissa thereby giving more weight to smaller values of v/[S]. Therefore, the typical measure of goodness
of fit for linear regression, the correlation coefficient R, is not applicable.
 HANES-WOOLF PLOT

A Hanes–Woolf plot is a graphical representation of enzyme kinetics in which the ratio of the initial
substrate concentration [S] to the reaction velocity v is plotted against [S].