Enzyme Kinetics II

The heavy metal Mercury (Hg)

Last Lecture
we examined how an enzyme can be characterized kinetically
conducting reactions with fixed concentrations of substrate

determining rates of reaction (v) from these

plotting rate vs [S] to yield Michaelis-Menten curve

This Lecture
we will examine a linear transformation of the M-M plot
use these curves to examine the effects of inhibitors on enzyme activity

Enzyme + cellulose Enzyme + glucose

y=mx+b

[glucose]

m=d[glucose]/dt

The rate (v) is the increase

in glucose liberated
from cellulose over time
slope of the line = initial rate
or velocity (v) of the reaction

time

Determination of reaction rates at varying substrate concentrations

[S]

multiple progress curves

etc.

[glucose]

[S]=4
[S]=3
[S]=2
[S]=1
time

Next, plot Rates vs [S]

1
2
3
4
5
6

v
0.001
0.002
0.004
0.007
0.010
0.013

Plotting substrate concentration vs rate The Michaelis-Menten curve

a rectangular hyperbola

v=

Vmax[S]
KM + [S]

rate, v
o
o
o
o

Vmax

o
o
o

Vmax

o
o
o
o
o

[substrate], [S]

KM
Problem: Vmax can only be estimated from the curve

Transformation of the Michaelis-Menten Equation: Lineweaver-Burk Equation

the Lineweaver-Burk transformation involves taking the reciprocal of
the M-M equation
v=

Vmax[S]
KM + [S]

1
v
1
v

the Lineweaver-Burk Equation

1
v
1
v

Notice this is now in the form of

the equation for a line:
y=mx + b

KM + [S]
=

Vmax[S]
KM

[S]

Vmax[S]

KM
=

Vmax[S]
KM

Vmax[S]

Vmax [S]

Vmax
1

Vmax

y = m x + b

this transformation is sometimes

called a double-reciprocal plot

1
v

slope =

1
KM

KM
Vmax

1
Vmax
1
[S]

1
v

KM
=

Vmax [S]

1
+

Vmax

y = m x + b

[S]
1
2
3
4
5
6

v
0.001
0.002
0.004
0.007
0.010
0.013

Michaelis-Menten Plot

Lineweaver-Burk Plot

 The Lineweaver-Burk* transformation was originally devised because it is

difficult to ascertain the actual value of Vmax from a Michaelis-Menten
plot
now however computer programs can be used to fit kinetic data to the equation
for a rectangular hyperbola and thus solve precisely for Vmax and KM
therefore, Lineweaver-Burk transformations are rarely used for their original
purpose
however, they are still qualitatively useful for examining the effect that inhibitors
have on the Vmax and KM characteristics of enzymes
Now we will consider enzyme inhibitors and use Lineweaver-Burk plots to
examine their effect on enzyme activities

* Eadie-Hofstee and Hanes-Woolf plots are two other common transformations

Molecules that inhibit enzymes are very important

they regulate enzyme activity in the body
but are also found in many other situations
Poisons
e.g. cyanide
Toxins
e.g. from poisonous mushrooms
Therapeutic Drugs
e.g. Antibiotics
e.g. Viagara
e.g. Ibuprofen (inhibitor of enzyme called Cyclooxygenase (COX))

There are 3 main types of enzyme inhibition mechanisms

inhibition is caused by molecules that bind enzymes and alter
either the Vmax or KM of the enzyme

Competitive Inhibition

occurs when a molecule structurally mimics the

substrate and binds the active site.
blocks (or competes with) the substrate for
binding to the active site

Note the slope of L-B plot is changed

since slope = KM/Vmax, one of these values must have
changed
Note that the y-intercept (1/Vmax) has not changed
therefore Vmax has not been altered

Note that it is the apparent Km that has been altered

(the x intercept = -1/KM)
so the apparent KM has been increased (note that the
real affinity of enzyme for substrate has not been altered
-just that more substrate needs to be added to compete
with inhibitor and saturate the enzyme
adding more substrate will outcompete the inhibitor and
the Vmax can be attained

Example of a Competitive Inhibitor

adenosine

Adenosine regulates sleep:

upon prolonged wakefulness, adenosine levels rise and promote
sleepiness by interacting with neuron receptors

Caffeine, which is a structural mimic (analogue) of adenosine, blocks adenosine

function by binding to the same neuronal receptors (enzymes).

Uncompetitive Inhibition
the inhibitor does not bind the active site, but instead
binds the ES, the enzyme-substrate complex, and prevents
the conversion of substrate to product

Note the slope of L-B plot remains the same, therefore

either there is no effect on either Km or Vmax (which
would mean that there is no inhibition) OR both have
been changed.
Note that the y intercept is increased, therefore Vmax
has been decreased AND the x-intercept has increased
therefore Km has decreased
so this inhibition primarily results from the fact that the
maximal velocity of the enzyme is severely decreased unlike competitive inhibition, this cannot be rescued by
adding more substrate
the decrease in Km (which should mean the enzyme now
has a greater affinity for substrate) results from fact that
the equilibrium shifts towards formation of more ES complex
as more inhibitor binds.

k-2

k-1

Effects of Uncompetitive Inhibitors on KM and Vmax

reaction rates are always measured

as initial
velocities,
before there
is breakdown
KM is actually aapproximation
combined rate constant that
describes
the formation
and
appreciable depletion of substrate
of the ES complex
or formation of product
fast

E+S

k1 k 1

slow

k2

ES

(k
k-1-1 + k2)

1
KE
M

+ Por

KM =

(k-1 + k2)

k1

k1

(k-1 + k2)

1
KM

in most reactions, k2 is usually much lower than k1

therefore KM approximately reduces to:

KM =

k-1
k1

since all enzyme is progressively trapped in the ES form, this mimics

an increase in k1 rate constant thus decrease in KM

when enzyme is saturated with substrate then the enzyme is operating as fast
as it can and Vmax = k2[ET] and
Vmax

k2 =

[ET]

= kcat

since the ES complex is trapped by the inhibitor, k2/kcat becomes very low and
Vmax is depressed and cannot be rescued by more substrate.

Noncompetitive Inhibition
here the inhibitor neither binds to the active site
nor binds the Enzyme-Substrate complex but rather
binds to a different site on enzyme.
this is the opposite of competitive inhibition in that
it is the Vmax that is altered (decreased) where as the Km
remains unchanged.
this means that the enzyme still has the same affinity
for the substrate, but the inhibitor negatively affects
the conversion of substrate to product
whenever the Vmax is affected, the kcat parameter will also
be affected since kcat = Vmax/ET

E+S

fast

slow

k1

k2

k-1

ES

E+P

non-competitive inhibition also cannot be rescued by

increases in [S]

Examples of Non-competitive Inhibitors

Heavy Metals like Lead (Pb), Mercury (Hg), Silver (Ag), Chromium (Cr)
Two effects
1) Interference with co-factor and co-enzyme function

Ferrochelatase (an enzyme) catalyzes

joining of protoporphyrin and Fe2+ to
form heme. Pb inhibits ferrochelatase.

2) Heavy metals disrupt disulfide bond formation in proteins by binding Sulfur atoms

Jim Clark 2007

Inhibitor Type

Effect

Competitive

Increased KM
Vmax unchanged

Uncompetitive

Decreased Vmax
Decreased KM
Ratio of KM/Vmax unchanged
kcat decreased

Noncompetitive

Decreased Vmax
KM unchanged
kcat decreased

Example of common Enzyme Inhibitors

Voltage-gated Na channels regulate Na+ ion passage across
axon membranes. Ingredients in insecticides (e.g. pyrethroids
like permethrin) trap the channel in an open conformation,
preventing repolarization of the membrane.

permethrin

the most common herbicide in the world

RoundUp Glyphosphate

analog of phosphoenolpyruvate, binds to EPSP synthase.

tyrosine
precursor for phenylalanine
tryptophan
synthesis

cyanide and carbon monoxide both bind heme co-factors and inhibit the
enzyme complex Cytochrome c oxidase

structure of cyanide anion

November 18, 1978: 909 people die from consuming cyanide in Jonestown
Guyana at the behest of a charismatic religious leader, Jim Jones.

Amanitin
a cyclic 8 amino acid peptide produced by
several genera of mushrooms
binds and inhibits RNA polymerase
reduces mRNA synthesis from a rate of
3000 nt/min to about 30 nt/min.

Aspirin
acetylsalicylic acid

Ibuprofen

Inhibit the COX (cyclooxygenase) enzymes that

control release of chemicals that mediate sensation
of pain

Structure of COX-1

Viagara (Sildenafil citrate)

inhibits an enzyme called cGMP-specific phosphodiesterase which regulates
blood vessel dilation and blood flow in tissue.
Originally tested as a treatment for reduced blood flow in heart muscle which
can lead to angina. It didnt work well but volunteers reported unusual side effects
during the clinical trials. The pharmaceutical company (Pfizer) took note.