Enzimas 2015 01 BqaBasica

ENZYMES
BIOQUIMICA BASICA
Sem 01/2015
Claudia Rubiano
Overview
1
Part I: Introduction to enzymes
Part II: Enzyme kinetics

Michaelis & Menten Kinetics
Allosteric enzymes kinetics
Inhibition of Enzyme Activity

Regulation of enzyme activity
Coenzymes
Defective enzymes and disease
Catalytic RNA


Coenzymes
Catalytic RNA
KEY CONCEPTS
Enzymes differ from simple chemical
catalysis in their efficiency and specificity.
An enzyme s name may reflect the
reaction it catalyzes.
There are three ways to increase the

rate of a chemical reaction.
Increasing the temperature
Adding energy in the form of heat
Increasing the concentration of the

reacting substances
Adding a catalyst
A substance that participates in the reaction
yet emerges from the reaction in its original
form
Enzymes are catalysts.

An enzyme is usually a protein that acts
as a catalyst to speed up the rate of a
chemical reaction without being consumed
itself.
Major exception: ribozymes are RNA
molecules that act as catalysts.
Enzymes speed up biochemical

reactions.
Rate enhancements of 108 to 1012 are typical of enzymes.
Enzymes have an active site.

Substrates bind to
enzymes at the active
site .
Serine proteases are an
exemplary class of
enzymes that have a
common set of amino
acids (red) in their active
site.
Most enzymes are highly specific for

their substrates.
Chymotrypsin has broad
substrate specificity,
though.
Chymotrypsin
Hydrolyzes peptide bonds
after Phe, Tyr, or Trp
Hydrolyzes other amide or
ester bonds after Phe, Tyr
or Trp
Enzymes are usually named after the

reaction they catalyze.
Pyruvate decarboxylase removes a
carboxylate group from the substrate
pyruvate.
There are six major classifications of

enzymes.
Classification is based on
the type of reaction
catalyzed by the enzyme.
What is the enzyme classification for

pyruvate decarboxylase?
Answer: Lyase
A CO2 group is eliminated
and a double bond is formed
to produce CO2.
For more info: see http://enzyme.expasy.org
KEY CONCEPTS
The height of the activation energy barrier
determines the rate of a reaction.
An enzyme provides a lower-energy
pathway from reactants to products.
Enzymes accelerate chemical reactions
using acid-base catalysis, covalent
catalysis, and metal ion catalysis.
The height of the activation energy

barrier determines the rate of a
reaction.
The higher the

activation energy
barrier,
the less likely
the reaction is to occur!
Products
The sign of !G indicates the

spontaneity of a reaction.
!G<0
spontaneous reaction
!G>0
non-spontaneous reaction
Enzymes work by lowering the

activation energy for a reaction.
Enzymes often use cofactors to aid

in catalysis.
There are three fundamental

mechanisms for enzyme catalysis.
Acid-base
Note: an enzyme can use acid catalysis, base
catalysis or both!
Covalent catalysis
Also called nucleophilic catalysis
Metal ion catalysis
Some amino acids can play a role in

acid-base catalysis.
Covalent Catalysis
Covalent catalysts accelerate reactions by
forming a covalent bond between E and S.
Nucleophiles & Electrophiles

Electron pair donor or
negative charge
Electron-deficient
atoms
Metal Ions as Catalysts
Example: Alcohol Dehydrogenase


Coenzymes
Catalytic RNA
KEY CONCEPTS
An enzymes activity, measured as the
rate of product formation, varies with the
substrate concentration.
Rates of Chemical Reactions

Enzyme kinetics is the study of rates of
reactions catalyzed by enzymes.
v = S
The reaction rate (velocity, v) can be

described in several ways.
Disappearance of substrate, S
Appearance of product, P
These equations relate velocity to

concentration of reactants and products.
Consider the reaction catalyzed by

triose phosphate isomerase.
As [GAP] decreases, the [DHAP]
increases.
GAP
DHAP
Reaction velocity can be thought of

as concentration vs. time.
Many enzymes react with substrates

in a nonlinear fashion.
The shape here
is hyperbolic.
Shape indicates,
in part, that E and
S combine to
form an ES
complex
E + S
ES


Coenzymes
Catalytic RNA
KEY CONCEPTS
Simple chemical reactions are described
in terms of rate constants.
The Michaelis-Menten equation describes
enzyme-catalyzed reactions in terms of
KM and Vmax.
Rate equations describe chemical

processes.
A unimolecular reaction has a velocity
(rate) that is dependent on the
concentration of only one substrate.
k
v = A
v = k [A], where k has units of sec-1
Rate equations describe chemical

processes.
A bimolecular (second order) reaction
has a velocity (rate) that is dependent on
two substrate concentrations.
k
v = A + B
v = k [A] [B] (or k [A]2 or k [B]2)

where k has units of M-1 ! sec-1
Many enzymes obey Michaelis-Menten

kinetics.
k1
k2
E + S ES
k-1
E+P
Rate limiting step

Problem:
[ES] is difficult to measure!
What can we do?
Try to re-express the rate.

k1
k2
E + S ES
k-1
E+P
One point that helps:

k1 [E] [S] - k-1 [ES] - k2 [ES]
Formation
of ES
Depletion of ES
Assume steady state equilibrium.

For most of the duration of the reaction,
[ES] remains steady as substrate is
converted to product.
Michaelis-Menten Equation
k1
k2
E + S ES
k-1
E+P
= k1 [E] [S] - k-1 [ES] - k2 [ES]
Derivation of the
= k1 [E] [S] - k-1 [ES] - k2 [ES]
Thus:
Since!
k1 [E] [S] = [ES] (k-1 + k2 )

[E]total = [ES] + [E]
[E] = [E]total - [ES]
Substitute
here
Derivation of the
k1 ([E]total [S] - [ES] [S]) = [ES] (k-1 + k2 )
Divide both sides by k1
KM
[E]total [S] - [ES] [S] = [ES] (k-1 + k2 )

k1
[E]total [S] - [ES] [S] = [ES] KM
Derivation of the
[E]total [S] - [ES] [S] = [ES] KM
Rearrange:
[E]total [S] = [ES] (KM + [S])
[ES] = [E]total [S]
KM + [S]
Derivation of the
Vmax
v =
The MichaelisMenten Equation
v =
k2 [E]total [S]
KM + [S]
Vmax [S]
KM + [S]
The Michaelis-Menten equation is

hyperbolic.
Vmax is where the reaction velocity reaches its plateau
KM is the substrate
concentration at ! Vmax
KEY CONCEPTS
The kinetic parameters KM , kcat, and
kcat/KM are experimentally determined.
K0.5 and Vmax values can be derived for
enzymes that do not follow the MichaelisMenten model.
Kinetic parameters are used to

compare enzyme activities
The Km can vary greatly from enzyme to enzyme, and
even for different substrates of the same enzyme:
Kinetic parameters are used to

compare enzyme activities
Considerations about Km and Vmax meaning:
They provide little information about the number,
rates, or chemical nature of discrete steps in the
reaction.
ONLY UNDER CERTAIN CONSIDERATIONS,
Km can be taken as a measure of the affinity of
an enzyme for its substrate in the complex ES
Comparing Catalytic Mechanisms

and Efficiencies
Kcat or turnover number: It is equivalent to the number of
substrate molecules converted to product in a given unit of
time on a single enzyme molecule when the enzyme is
saturated with substrate.
The catalytic rate constant determines

how quickly an enzyme can act.
kcat = catalytic rate
constant, turnover
kcat = k2
kcat = Vmax/[E]total

and Efficiencies
Kcat and Km allow to evaluate the kinetic efficiency of enzymes, but either
parameter alone is insufficient for this task.
The best way to compare the catalytic efficiencies of different enzymes
or the turnover of different substrates by the same enzyme is to compare
the ratio Kcat/Km for the two reactions: Specificity constant

and Efficiencies
Kcat and Km allow to evaluate the kinetic efficiency of enzymes, but either
parameter alone is insufficient for this task.
The best way to compare the catalytic efficiencies of different enzymes
or the turnover of different substrates by the same enzyme is to compare
the ratio Kcat/Km for the two reactions: Specificity constant
kcat/KM indicates catalytic efficiency.

What limits the catalytic power of an enzyme?
Electronic rearrangements during formation of the
transition state
Frequency of productive enzyme collision with
substrate, with the maximum being the diffusioncontrolled limit
Enzymes reach catalytic perfection when their
rate is diffusion-controlled.
There are algebraical

transformations of the MM equation
into versions that are useful in the
practical determination of Km and
Vmax
The Lineweaver-Burk plot

v =
Vmax [S]
KM + [S]
Take the reciprocal of both sides:
1
v
KM
Vmax
[S]
1
Vmax
The Lineweaver-Burk plot

Notice how the data are weighted heavily
here due to the linearization!
The EadieHofstee plot
The Hanes-Woolf plot
Many enzymes catalyze reactions with two or more

substrates
ATP + glucose ADP +glucose 6-phosphate
The rates of bisubstrate reactions can also be analyzed by the

Michaelis-Menten approach.
There are dierent possible mechanisms:
Mech
How to distinguish between these possibilities?

Steady-state kinetics can often help View

substrates

Mech


substrates

Mech


substrates

Mech

Not all enzymes fit the MichaelisMenten model.

Some enzymes have multiple substrates.

Some enzyme-catalyzed reactions have
many steps.
With allosteric enzymes, binding of a substrate

at one active site can affect the catalytic activity
of other active sites.
Allosteric enzymes exhibit cooperativity.

Velocity plot is sigmoidal!


Coenzymes
Catalytic RNA

Coenzymes
Catalytic RNA

Isosteric and allosteric enzymes
One Vs Various conformations

Allosteric Enzymes: dierent catalytic properties, the
proportion of the total number of enzyme molecules is
influenced by substrates and other ligands
There are dierences in kinetics: the anity of allosteric
enzymes is not constant, but depends on the type and
concentration of the ligand. View

Coenzymes
Catalytic RNA



Coenzymes
Catalytic RNA



Coenzymes
Catalytic RNA


Allosteric enzymes
Control [regulatory] enzymes
Have quaternary structure
Have active site and modulatory site
* Active site binds substrate to give product
* Modulatory site binds +ve or -ve modulator to
increase or decrease the activity of the active site
Catalyse an irreversible reaction
Inhibited by end product: Feedback inhibition
Activated by substrate and other positive
modulators
Do not obey Michaelis Menten Kinetics
Allosteric Enzyme example: PFK


Coenzymes
Catalytic RNA
KEY CONCEPTS
Noncompetitive, mixed, and
uncompetitive inhibitors decrease kcat.
Allosteric regulators can inhibit or activate
enzymes.
Some inhibitors act irreversibly.
A suicide inhibitor
Irreversible Inhibitors
E + I EI
E doesnt regain activity
E-I bind in a covalent way
Suicide inhibition: occurs when an enzyme binds a substrate

analogue and forms an irreversible complex with it through a
covalent bond during the normal catalysis reaction.
E + I EI

E + I EI

E + I EI

Reversible inhibitors
E + I EI
* E & I bind by non-covalent bonds
* E I can be dissociated by: dilution, dialysis
1
Competitive
Uncompetitive
Mixed (non-competitive)
E + I EI
1
Competitive
Uncompetitive
E + I EI
1
Competitive
Uncompetitive
E + I EI
1
Competitive
Uncompetitive
Competitive inhibition
Competitive inhibitors are generally substrate or transition

state analogues
They have properties similar to those of one of the substrates
(or transition state) of the target enzyme: competition S vs I
for active site
Km increases but Vmax does not change
Can be reverted by high substrate concentration
Ki =
[E ][I ]
[I ]
; Kmapp = Km (1 + )
[EI ]
Ki
Uncompetitive inhibition
Inhibitor binds only to the ES complex and apparently distorts

the active site
Vmax and Km decrease
Kiu =
app
Vmax
[ES][I ]
[ESI ]
Vmax
Km
=
; Kmapp =
[I ]
[I ]
(1 +
)
(1 +
)
Kiu
Kiu
Vmax
is unaected
Km
Mixed inhibition (non-competitive)
Inhibitor binds to both the enzyme and the enzymesubstrate

complex and may interfere with both substrate binding and
catalysis
The apparent Vmax decreases and the Km apparent may
decrease or increase
When only Vmax is aected, the inhibition is said to be
non-competitive simple or pure
[E ][I ]
[ES][I ]
; Ki =
[EI ]
[ESI ]
V
max
app
Vmax
=
[I ]
(1 + )
Ki
Ki =
Kinetics of Inhibition
Enzymatic Inhibition: Summary
Use of enzyme inhibitors
As drugs (in pharmacology)

Antiviral, Antibacterial, Anti tumor drugs
Agricultural pesticides and insecticides
Study of enzyme mechanisms of action
Diagnoses of diseases.
In prostate cancer acid phosphatase is increased, L-tartrate
inhibits competitively 95% of the acid phosphate from
prostrate, but has lower inhibitory eect on acid phosphatase
from other sources. Samples from suspected carcinoma
patients can be assessed in presence and absence of L-tartrate.
Example: Sulfa drugs
Example: Methotrexate
Structural analog of folic acid competes with dihydro-folate
reductase.It is used for treatment of leukaemia.
Commonly Used Drugs that are Enzyme Inhibitors


Coenzymes
Catalytic RNA

Coenzymes
Catalytic RNA
Mechanisms of regulation
Temperature, pH, cofactors, coenzymes, range of substrates

and location all help regulate enzymes.
pH eect
Examples of optimum pH
Enzyme
Source
pepsin
sucrase
catalase
arginase
alkaline phosphatase
gastric mucosa
intestine
liver
beef liver
bone
Optimum pH
1.5
6.2
7.3
9.0
9.5
Temperature eect

Coenzymes
Catalytic RNA
Mechanisms of regulation
Several other methods are available to the cell for regulation:

Long term (hours or more): protein synthesis
Short term (seconds): changes in catalytic eciency
* Feed back inhibition: a kind of allosteric regulation
* Regulatory covalent modifications: reversible or irreversible
Feed back inhibition
Irreversible modifications: proteolysis
Certain enzymes are synthesized and secreted as inactive

precursor proteins known as proenzymes or zymogens
Proenzymes facilitate rapid mobilization of an activity in
response to physiologic demand
Ex: digestive enzymes pepsin, trypsin, and chymotrypsin
(proenzymes = pepsinogen, trypsinogen, and
chymotrypsinogen, respectively) Prot
Irreversible modifications: proteolysis
Certain enzymes are synthesized and secreted as inactive

precursor proteins known as proenzymes or zymogens
Proenzymes facilitate rapid mobilization of an activity in
response to physiologic demand
Ex: digestive enzymes pepsin, trypsin, and chymotrypsin
(proenzymes = pepsinogen, trypsinogen, and
chymotrypsinogen, respectively) Prot
Reversible covalent modifications

Phosphorylation-dephosphorylation: Protein Kinases and
phospatases
Involves several proteins and ATP and is under direct neural
and hormonal contro


Coenzymes
Catalytic RNA
Coenzymes
In many ez-catalyzed reactions, electrons or groups of atoms are
transferred from one substrate to another. This type of reaction
always also involves additional molecules (coenzymes), which
temporarily accept the group being transferred.
Coenzymes can be soluble (S) or act as a prostetic group (P).
Coez are chemically changed by the enzymatic reactions in which
they participate: in order to complete the catalytic cycle, the
coenzyme must be returned to its original state.
For prosthetic groups, this can occur only in a separate phase of the
enzymatic reaction sequence. For transiently bound coenzymes,
such as NAD, the regeneration reaction may be catalyzed by a
dierent enzyme.
Redox coenzymes and Group-transferring coenzymes


Coenzymes
Catalytic RNA
Some examples
A number of hereditary diseases result from the absence of an

enzyme or a defective one.

Coenzymes
Catalytic RNA
Phenylketonuria (PKU)
Genetic defect that results in a defect of the enzyme
phenylalanine hydroxylase.
Aects about 1 baby per 13,000.
Children are screened at birth.
Can result in retarded physical and mental development if
untreated.
Treatment: restrict phenylalanine until age 10 (until brain is
developed).
PKU is one of a family of enzymatic/genetic disorders related
to phenylalanine metabolism.
Phenylketonuria (PKU)


Coenzymes
Catalytic RNA
Catalytic RNA
Since their discovery, it was believed that all enzymes were

proteins.
In 1981 - 1982, two research group reported results on
catalytic RNA.
In 1989, the Nobel Prize in Chemistry was awarded to Sidney
Altman (Yale) and Thomas Cech (University of Colorado Boulder) for their discovery.
The term ribozyme is now used for RNA enzymes.
Ribonuclease P
This was the first type of catalytic RNA discovered and is present
in all organisms
Substrates are at least 60 inactive, precursor forms of tRNA.
Ribonuclease P acts to remove a segment of the
ribonucleotide, producing mature tRNA.
The enzyme consists of a small protein subunit with a
molecular weight of 14,000 and an RNA component of 377
nucleotides.

Coenzymes
Catalytic RNA

Coenzymes
Catalytic RNA
Significance of ribozymes
Other forms of catalytic RNA continue to be discovered.

Small ribozymes have been found as components of plant
RNA viruses.
The active region of this RNA consists of only 19 - 30
nucleotides.
Because of their characteristic shape and action, they are
called hammerhead ribozymes.

Coenzymes
Catalytic RNA

Coenzymes
Catalytic RNA
go back
go back

Coenzymes
Catalytic RNA
go back
go back

Coenzymes
Catalytic RNA
go back
go back

Coenzymes
Catalytic RNA
go back

Coenzymes
Catalytic RNA
go back

Coenzymes
Catalytic RNA
go back
Proteolysis example
go back
Proteolysis example
go back

Enzimas 2015 01 BqaBasica

Încărcat de

Informații document

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Enzimas 2015 01 BqaBasica

Încărcat de

Drepturi de autor:

Formate disponibile

ENZYMES

Part I: Introduction to enzymes

Part II: Enzyme kinetics

Inhibition of Enzyme Activity

Defective enzymes and disease

Part I: Introduction to enzymes

Part II: Enzyme kinetics

Inhibition of Enzyme Activity

Defective enzymes and disease

There are three ways to increase the

Increasing the concentration of the

Enzymes are catalysts.

Enzymes speed up biochemical

Rate enhancements of 108 to 1012 are typical of enzymes.

Enzymes have an active site.

Most enzymes are highly specific for

Enzymes are usually named after the

There are six major classifications of

What is the enzyme classification for

The height of the activation energy

The higher the

The sign of !G indicates the

Enzymes work by lowering the

Enzymes often use cofactors to aid

There are three fundamental

Metal ion catalysis

Some amino acids can play a role in

Nucleophiles & Electrophiles

Metal Ions as Catalysts

Example: Alcohol Dehydrogenase

Part I: Introduction to enzymes

Part II: Enzyme kinetics

Inhibition of Enzyme Activity

Defective enzymes and disease

Rates of Chemical Reactions

The reaction rate (velocity, v) can be

These equations relate velocity to

Consider the reaction catalyzed by

Reaction velocity can be thought of

Many enzymes react with substrates

Part I: Introduction to enzymes

Part II: Enzyme kinetics

Inhibition of Enzyme Activity

Defective enzymes and disease

Rate equations describe chemical

v = k [A], where k has units of sec-1

Rate equations describe chemical

v = k [A] [B] (or k [A]2 or k [B]2)

Many enzymes obey Michaelis-Menten

Rate limiting step

Try to re-express the rate.

One point that helps:

Assume steady state equilibrium.

= k1 [E] [S] - k-1 [ES] - k2 [ES]

k1 [E] [S] = [ES] (k-1 + k2 )

[E]total [S] - [ES] [S] = [ES] (k-1 + k2 )

The MichaelisMenten Equation

The Michaelis-Menten equation is

Kinetic parameters are used to

Kinetic parameters are used to

Comparing Catalytic Mechanisms

The catalytic rate constant determines

Comparing Catalytic Mechanisms

Comparing Catalytic Mechanisms