ENZYME INHIBITION

AND DEACTIVATION

Lecture 14-15
 OUTLINE
 enzyme inhibition- competitive, non-competitive
and un-competitive
 INTRODUCTION
 The activity of many enzymes can be inhibited by
the binding of specific small molecules and ions.
This means of inhibiting enzyme activity serves as
a major control mechanism in biological systems.
 Enzyme inhibition refers to the decrease
of enzyme-related processes.
 In enzyme activity, inhibition refers to the
decrease of an enzyme's activity, caused by a
substance called an enzyme inhibitor
 Many substances alter the activity of an enzyme by
combining with it in a way that influences the
binding of substrate and/or its turnover number.
Substances that reduce an enzyme’s activity in this
way are known as inhibitors.

 Enzyme inhibitors are molecules that interfere

with catalysis, slowing or halting enzymatic
reactions.

 There are two broad classes of enzyme inhibitors:

 Reversible and
 Irreversible.
 An irreversible inhibitor dissociates very slowly
from its target enzyme because it has become
tightly bound to the enzyme, either covalently or
noncovalently. Some irreversible inhibitors are
important drugs.
 Penicillin acts by covalently modifying the enzyme
transpeptidase, thereby preventing the synthesis of
bacterial cell walls and thus killing the bacteria
 Reversible inhibition, in contrast with irreversible
inhibition, is characterized by a rapid dissociation
of the enzyme-inhibitor complex.
 Reversible enzyme inhibitors diminish an enzyme’s
activity by interacting reversibly with it
 Reversible Inhibition can be classified as

 Competitive,

 Un-competitive inhibition

 Non-competitive or Mixed Inhibition and

 COMPETITIVE INHIBITION

 Competitive Inhibition Involves Inhibitor Binding

at an Enzyme’s Substrate Binding Site.
 A substance that competes directly with a normal
substrate for an enzyme’s substrate-binding site is
known as a competitive inhibitor.
 Such an inhibitor usually resembles the substrate
so that it specifically binds to the active site but
differs from the substrate so that it cannot react as
the substrate does.
 For example, succinate dehydrogenase, a citric
acid cycle enzyme that converts succinate to
fumarate, is competitively inhibited by
malonate, which structurally resembles succinate
but cannot be dehydrogenated:
 The Degree of Competitive Inhibition Varies with
the Fraction of Enzyme That Has Bound Inhibitor.
 The general model for competitive inhibition is
given by the following reaction scheme:
 Here, I is the inhibitor, EI is the catalytically
inactive enzyme–inhibitor complex, and it is
assumed that the inhibitor binds reversibly to the
enzyme and is in rapid equilibrium with it so that

 A competitive inhibitor therefore reduces the

concentration of free enzyme available for
substrate binding.
 In the presence of a competitive inhibitor, the
Michaelis- Menten equation becomes
 Because the inhibitor binds reversibly to the
enzyme, the competition can be biased to favor
the substrate simply by adding more substrate.
 When [S] far exceeds [I], the probability that an
inhibitor molecule will bind to the enzyme is
minimized and the reaction exhibits a normal
Vmax.
 Km, increases in the presence of inhibitor by the
factor α.
 Practice question

 Write down the properties of compititive

inhibition.
 Explain the following figure
UNCOMPETITIVE INHIBITION
 Uncompetitive Inhibition Involves Inhibitor
Binding to the Enzyme–Substrate Complex
 In uncompetitive inhibition, the inhibitor binds
directly to the enzyme– substrate complex but not
to the free enzyme:
 The binding of uncompetitive inhibitor, which
need not resemble substrate, presumably distorts
the active site, thereby rendering the enzyme
catalytically inactive.
 In the presence of an uncompetitive inhibitor, the
Michaelis-Menten equation is altered to

 at high concentrations of substrate, V0 approaches

Vmax/α’. Thus, an uncompetitive inhibitor lowers
the measured Vmax
 Apparent Km also decreases, because the [S]
required to reach one-half Vmax decreases by the
factor α’.
 Practice question

 Write down the properties of uncompititive

inhibition.
EXPLAIN THE FOLLOWING FIGURE
 MIXED INHIBITION
 Mixed Inhibition Involves Inhibitor Binding to
Both the Free Enzyme and the Enzyme–Substrate
Complex
 A mixed inhibitor also binds at a site distinct from
the substrate active site, but it binds to either E or
ES.
 Many reversible inhibitors interact with the enzyme
in a way that affects substrate binding as well as
catalytic activity. In other words, both the enzyme
and the enzyme–substrate complex bind inhibitor,
resulting in the following model:
 This phenomenon is known as mixed inhibition
(alternatively, noncompetitive inhibition).
Presumably, a mixed inhibitor binds to enzyme
sites that participate in both substrate binding
and catalysis.
 For example, metal ions, which do not compete
directly with substrates for binding to an enzyme
active site, as do competitive inhibitors, may act
as mixed inhibitors. The two dissociation constants
for inhibitor binding
 The rate equation describing mixed inhibition is

 where α and α’ are defined before.

 A mixed inhibitor usually affects both Km and Vmax. The
effect of a noncompetitive inhibitor is to reduce [ES] that
can advance to product .Since Vmax = k2[Et], and the
concentration of competent Et is diminished by the
amount of ESI formed
 Vmax is decreased
 mixed inhibition are observed only for enzymes with two or
more substrates
 Practice question

 Write down the properties of noncompititive

inhibition.
 This form of the Michaelis-Menten equation is
called the Lineweaver-Burk equation.
LINEWEAVER-BURK EQUATION FOR
INHIBITION
CALCULATIONS
 Computer-assisted design of compounds that fit snugly into
an enzyme’s active site is an essential part of modern drug
development.
 For example, the anti-influenza drug oseltamivir (Tamiflu)
is hydrolyzed in the liver (Fig) to yield oseltamivir
carboxylate, a competitive inhibitor of influenza
neuraminidase that binds to this enzyme’s active site.
 Neuraminidase catalyzes the hydrolysis of neuraminic acid
(sialic acid) to help the viral particles escape from the host
cell surface, where membrane glycoproteins contain sialic
acid residues.
 Oseltamivir carboxylate, which can significantly limit the
production of the flu virus
ENZYME INHIBITOR AS DRUG
 Penicillin Irreversibly Inactivates a Key Enzyme
in Bacterial Cell-Wall Synthesis
 Penicillin, the first antibiotic discovered, consists
of a thiazolidine ring fused to a b-lactam ring, to
which a variable R group is attached by a peptide
bond. In benzyl penicillin, for example, R is a
benzyl group
 penicillin was inferred to interfere with the
synthesis of the bacterial cell wall. The cell-wall
macromolecule, called a peptidoglycan, consists of
linear polysaccharide chains that are cross-linked
by short peptides.
 The enormous bag-shaped peptidoglycan confers
mechanical support and prevents bacteria from
bursting in response to their high internal osmotic
pressure
 In 1965, James Park and Jack Strominger
independently deduced that penicillin blocks the
last step in cell-wall synthesis namely, the
crosslinking of different peptidoglycan strands.
 In the formation of the cell wall of Staphylococcus
aureus, the amino group at one end of a
pentaglycine chain attacks the peptide bond
between two D-alanine residues in another peptide
unit
 A peptide bond is formed between glycine and one
of the D-alanine residues; the other D-alanine
residue is released. This cross-linking reaction is
catalyzed by glycopeptide transpeptidase.
 Penicillin is welcomed into the active site of the
transpeptidase because it mimics the D-Ala-D-Ala
moiety of the normal substrate.
 Bound penicillin then forms a covalent bond with a
serine residue at the active site of the enzyme.
 This penicilloyl-enzyme does not react further.
Hence, the transpeptidase is irreversibly inhibited
and cell-wall synthesis cannot take place.
 DRUGS AGAINST HIV

 Several of the drugs used to block HIV protease

compounds that were designed to bind the enzyme
with high affinity
 HIV attaches to a target cell and injects its
genetic material into the host cell.
 RNA rather than DNA is genetic material here.
 The viral RNA is transcribed into DNA by a viral
enzyme called reverse transcriptase
 Most of the viral proteins are synthesized as parts
of larger polypeptide precursors known as p
 Consequently, proteolytic processing by the virally
encoded HIV protease to proteins. release these
viral proteins is necessary for viral reproduction
 In the absence of an effective vaccine for HIV,
efforts to prevent and treat AIDS have led to the
development of compounds that inhibit HIV
reverse transcriptase and HIV protease
 HIV protease is a homodimer of 99-residue subunits.
Mechanistically, it is a so-called aspartic protease, a
family of proteases that includes pepsin
 HIV protease cleaves a number of specific peptide
bonds, including Phe–Pro and Tyr–Pro
peptide bonds in its physiological substrates, the HIV
polyproteins
 The peptidomimetic(peptide-imitating) drugs
ritonavir and saquinavir contain phenyl and other
bulky groups that bind in the HIV protease active site
 Several inhibitors of reverse transcriptase
have been developed
 AZT (3ʹ-azido-3 ʹ-deoxythymidine;
Zidovudine)
 2 ʹ,3ʹ -dideoxycytidine (ddC, Zalcitabine)
 2 ʹ,3 ʹ -dideoxyinosine (ddI, Didanosine
 Nevirapine, Nonnucleoside compounds
 The archetype is AZT (3-azido-3-
deoxythymidine; Zidovudine), which is taken up
by cells, phosphorylated, and incorporated into
the DNA chains synthesized from the HIV template
by reverse transcriptase.
 Because AZT lacks a 3-OH group, it cannot support
further polynucleotide chain elongation and
therefore terminates DNA synthesis.
 Most cellular DNA polymerases have a low affinity
for phosphorylated AZT, but reverse transcriptase
has a high affinity for this drug, which makes AZT
effective against viral replication.
 Other nucleoside analogs that are used to treat
HIV infection are 2,3-dideoxycytidine (ddC,
Zalcitabine) and 2,3-dideoxyinosine (ddI,
Didanosine; inosine nucleotides are
metabolically converted to adenosine and
guanosine nucleotides), which also act as chain
terminators.
 Nonnucleoside compounds such as nevirapine
do not bind to the reverse transcriptase active
site but instead bind to a hydrophobic pocket
elsewhere on the enzyme.