BMB 506 PROTEINS AND ENZYMES (4)

Understand the importance of an ENZYME-SUBSTRATE complex in

catalysis.

Understand the meanings of Km and Vmax.

Know how to determine Km and Vmax from a Lineweaver-Burke plot.

Understand the difference between COMPETITIVE and NONCOMPETITIVE inhibition.

Be able to distinguish between competitive and non-competitive

inhibition from Lineweaver-Burke plots.

Know examples of competitive and non-competitive inhibitors.

ENZYMES CANNOT ALTER REACTION

EQUILIBRIA

Forward rate constant ( kF ) = 10-4 sec-1

Reverse rate constant ( kR ) = 10-6 sec-1
At equilibrium [B] / [A] = 100 in presence or absence of enzyme.
Enzymes reduce the time required to reach equilibrium.

TRANSITION STATE STABILIZATION

Enzymes accelerate reactions by decreasing the free
energy of activation.

ENZYME SUBSTRATE COMPLEX

The velocity of an enzyme catalyzed
reaction increases with increased
substrate concentration until a
maximum velocity is reached.
This observation was interpreted by
Leonor Michaelis in 1913 to indicate
that each enzyme molecule had a
finite number of catalytic sites at
which substrate molecules bound to
form an ENZYME SUBSTRATE
COMPLEX (ES).

ENZYME SUBSTRATE COMPLEX

Formation of an ES complex can
sometimes be detected
spectroscopically, as for the enzyme
Tryptophan synthetase.
TS catalyses the synthesis of Ltryptophan from L-serine and indole.
Fluorescence from a pyridoxal
phosphate prosthetic group on the
enzyme is altered upon substrate
binding.

THE MICHAELIS - MENTEN MODEL

Leonor Michaelis and Maud Menten proposed a model to account

for the observed relationship between the rate of an enzyme
catalyzed reaction (V) and the substrate concentration [S].
The critical feature of their proposed model is the requirement for
a specific enzyme-substrate complex ES as an intermediate in the
reaction.
k1
k3
E + S < ==> ES == > E + P
(1)
k2
E combines with S to form ES with a rate constant k1. ES can
either dissociate to E and S with a rate constant k2 or proceed to
form product with a rate constant k3.
It is assumed that E and P do not revert to ES. This is true initially
when [P] is very low.

MICHAELIS MENTEN EQUATION

V = Vmax

[S] << KM
[S] >> KM

V = [S] Vmax / KM
V = Vmax

[S] = KM

V = Vmax / 2

[S]
__________
[S] + KM

Rate proportional to [S]

Rate independent of [S]

Thus: KM is equal to the substrate concentration at which

the reaction velocity is half maximal.

LINEWEAVER BURKE PLOT

1 / V = 1 / Vmax + KM / Vmax ( 1 / [S] )

COMPETITIVE INHIBITION
Inhibitor competes with the substrate for the same binding site on
the enzyme.
Frequently the inhibitor is a chemical analogue of the substrate.

Vmax unchanged.
Apparent Km changes.
1 / V = 1 / Vmax + Km / Vmax
(1 + [I] / Ki) ( 1 / [S])

EXAMPLE OF COMPETITIVE INHIBITION

SUCCINATE DEHYDROGENASE

Typically competitive inhibitors are chemical analogues of the substrate.

Succinate dehydrogenase catalyses the dehydrogenation of succinate to fumarate.
Malonate has a similar chemical structure to succinate and can therefore bind in
the active site. However, when it binds to the enzyme there is no reaction.
Malonate is therefore a competitive inhibitor.
However, high concentrations of succinate can COMPETE with malonate and
thereby reduce or eliminate the inhibition.

NON-COMPETITIVE INHIBITION

The inhibitor binds at a site different from the substrate binding site.

Km unchanged
Vmax reduced

EXAMPLE OF NON-COMPETITIVE
INHIBITION: ACETYLCHOLINESTERASE
ACETYLCHOLINESTERASE:
Occurs in synapses of
neuromuscular junctions.
It catalyses the hydrolysis of the
neurotransmitter acetylcholine to
choline and acetate.
An active site Ser is essential for
catalysis.

SARIN NERVE GAS IS A NON-COMPETITIVE

INHIBITOR OF ACETYLCHOLINESTERASE
Organophosphonates phosphonylate
the active site SER of Achase,
inactivating the enzyme.
The nerve gas SARIN is one example
of this type of non-competitive
inhibitor of Achase.
A dose of 0.5 mg is lethal in humans.
Paralysis and DEATH result from
exposure to this nerve gas.
Metal ions such as Hg++ and Pb++ are
frequently non-competitive inhibitors
of enzymes having active site CYS
residues.

ACTIVE SITES OF ENZYMES

The Active site is relatively small region of an enzyme where the

substrates bind and amino acids that participate in catalysis are
located. Prosthetic groups such as metal ions or small molecules
that are required for catalysis are also located in the active site.

Active sites are 3-dimensional entities, frequently composed of

amino acids widely separated in the linear amino acid sequence.
E.g. Lysozyme residues 35, 52, 62 and 101 in a sequence of 129
residues.

Active sites are frequently clefts or crevices from which water may
be excluded. In multi-domain enzymes the active site is often found
at the interface between 2 domains.

ENZYME SUBSTRATE INTERACTIONS

Enzyme-substrate binding involves multiple weak forces
such as Van der Waals interactions, hydrogen bonds and
electrostatic interactions.
ES complexes usually have equilibrium constants ranging
from 10-2 to 10-8 M, corresponding to free energies of
interaction ranging from 3 to 12 kcal/mol.
Van der Waals interactions resulting from complementarity
in the shapes of the enzyme and substrate contribute to the
substrate specificities of enzymes.

SPECIFICITIES OF PROTEASES
TRYPSIN:
A substrate binding pocket in
the active site of in trypsin
contains a negatively charged
amino acid to interact with Arg
or Lys in the substrate.
CHYMOTRYPSIN:
The equivalent pocket in
chymotrypsin is hydrophobic
confering specificity for peptide
cleavage adjacent to bulky
hydrophobic residues such as
Phe or Ile.

LOCK AND KEY MODEL

As early as 1890, Emil Fischer likened the interaction of an
enzyme with its substrate to the fit of a key in a lock.

INDUCED-FIT MODEL

It is now known that conformational changes can accompany substrate

binding, such that complementarity in shape is only achieved after binding.
This idea was first proposed by Daniel Koshland Jr. in 1958 as the induced-fit
model of ES complex formation.

INDUCED FIT
Glucose binding to the enzyme Hexokinase promotes a large
conformational change.

COFACTORS
COFACTORS are small chemical groups located in the
active sites of some enzymes, and are required for catalytic
activity. Tightly bound organic cofactors are sometimes
termed prosthetic groups.
Many enzymes require metal ions such as Mn2+ (arginase),
Zn2+ (carboxypeptidase) or Mg2+ (hexokinase) as cofactors.
Other cofactors include larger molecules such as heme.

In the absence of a required cofactor the enzyme is termed

an APOENZYME and in its presence a HOLOENZYME.

CARBOXYPEPTIDASE A
The Zn2+ ion in carboxypeptidase promotes electron shift from
the C=O bond to the O- transition state.

METAL ION COFACTORS

COENZYMES

COENZYMES are sometimes referred to as co-substrates, because

one molecule of coenzyme is required for each molecule of
substrate converted to product. Coenzymes must be regenerated
by a different reaction.
Nicotine adenine dinucleotide (NAD) and its phosphorylated form
NADP are examples of coenzymes.
In some texts coenzymes are included under the general group of
cofactors but this definition is somewhat arbitrary.

NAD-BINDING DOMAINS IN
DEHYDROGENASES

The NAD binding domains of alcohol dehydrogenase (left) and

glyceraldehyde phosphate dehydrogenase (right) have similar
structures, but their substrate-binding domains are quite different.
Commonly, the substrate and coenzyme bind to different domains of
the enzyme.

COENZYMES ACT AS CARRIERS

Coenzymes frequently act as carriers of chemical groups. When bound

to the coenzyme the group carried exists in an activated form.