enzyme catalysis - genetics and bioengineering · function: hydrolysis of rna to component...
TRANSCRIPT
How do enzymes catalyze chemical
reactions?
Theoretical concept: transition state theory
Catalytic mechanisms:
• Acid-base catalysis
• Covalent catalysis
• Metal ion catalysis
• Electrostatic catalysis
• Catalysis through proximity and orientation effects
• Catalysis by preferential transition state binding
Example: Serine proteases
Making reactions go faster
Increasing the temperature makes molecules move faster
Biological systems are very sensitive to temperature changes.
Enzymes can increase the rate of reactions without
increasing the temperature.
They do this by lowering the activation energy.
They create a new reaction pathway “a short cut”
Transition state theory
Transition state diagrams illustrate the path of a reaction
The transition state - the point of highest energy, i.e. the
most unstable situation
ΔG‡ is the difference of energy of the reactants and the
transition state (free energy of activation)
ΔG‡ determines the rate of the reaction
The reaction rate is proportional to e- ΔG‡/RT
R is the gas constant and T the absolute temperature (Eyring
equation)
greater the value of ΔG‡, the slower the reaction rate
Acceleration of a reaction rate can therefore be achieved by
lowering ΔG‡
Enzymes reduce ΔG‡
Catalysts lower the energy of activation of a chemical
reaction and hence accelerate the rate of reaction
The rate of a chemical reaction can be accelerated by up to
1017-fold
The rate acceleration is enzyme specific and depends on the
nature of the chemical reaction.
a catalyst does not change the equilibrium constant
between reactants and products
Acid-base catalysis
A reaction is acid-catalyzed if proton donation (a Brønsted
acid) lowers the free energy of activation and leads to an
acceleration of the reaction rate.
A reaction is base-catalyzed if proton abstraction (a
Brønsted base) lowers the free energy of activation and leads
to an acceleration of the reaction rate.
Reactions catalyzed by the concerted action of a proton
donation and abstraction are called acid-base catalyzed.
RNAase A - example for an enzyme utilizing acid-
base catalysis
Function: Hydrolysis of RNA to component nucleotides
His12 abstracts a proton from the 2’-OH group.
This promotes the nucleophilic attack on the
phosphorus resulting in P-O bond cleavage.
His119 acts as a general acid by protonation of the
oxyanion leaving group. This results in the
formation of a 2’,3’-cyclic intermediate which can
be isolated under certain conditions. Water is not
admitted to the active site and drives essentially
the reversal of the initial process leading to
complete hydrolysis.
Covalent catalysis
Catalyst forms a transient covalent linkage with the substrate
leading to rate enhancement.
Can be divided into two phases:
1. Nucleophilic reaction between the catalyst and the substrate
to form a covalent bond
2. Withdrawal of electrons from the reaction center by the
electrophilic catalyst (e.g. hydrolysis of the Schiff base).
Note that in cases where the nucleophilicity is the rate-determining step, the
reaction rate tends to increase with the basicity (pK) of the catalyst.
Covalent catalysis
Top: uncatalyzed decarboxylation of acetoacetate
Bottom: primary amines as catalyst for the decarboxylation
of acetoacetate
Groups involved in covalent catalysis Among the amino acids, the following side chains can be used for
covalent catalysis:
• Serine: hydroxyl group (an example will follow later!)
• Cysteine: thiol group
• Histidine: imidazol ring
• Aspartate & glutamate: carboxyl group
• Lysine: amino group (Schiff base formation)
In addition, enzymes can utilize organic compounds, so called coenzymes, such as pyridoxal phosphate and thiamine pyrophosphate (vitamins!) for covalent catalysis.
Metal ion catalysis
One third of all enzymes require the presence of metal ions for activity!
1. Metalloenzymes - contain tightly bound metal ions, most
commonly transition metals such as iron, copper, manganese
and cobalt.
2. Metal-activated enzyme - bind metal ions loosely from
solution, most commonly alkali and alkaline earth metals such as
sodium, potassium, magnesium and calcium
Roles of metal ions in catalysis
• Bind to substrate in order to properly align it for catalysis
• Mediate oxidation-reduction processes (redox biochemistry)
through reversible interchange of the metal ion’s oxidation state.
• Stabilization/shielding of negative charges during the catalytic
process.
Metal ions promote nucleophilic catalysis
Metal-bound water exhibits a much lower pKa value
The resulting metalbound hydroxyl group can act as a potent
nucleophile
The essential zinc in the active site is bound by three
histidine side chains.
Water occupies the fourth binding site and is
polarized by the zinc atom.
The bound water deprotonates and the resulting
hydroxyl attacks carbon dioxide converting it to
carbonate.
Proximity and orientation effects
Binding of the substrate facilitates catalysis in three ways:
Proximity - effect is contributing a rate acceleration of ca.
5-fold
Orientation - Reaction rates are accelerated approx. 100-
fold by this effect
Freezing out motion - the enzyme restricts the
translational and vibrational freedom of the substrate an
„prepares“ it for a transition state like structure. The rate
enhancement achieved in the order of 107
The active site
For catalysis
The shape and the chemical environment inside the active
site permits a chemical reaction to proceed more easily
Cofactors
An additional non-protein molecule that is needed by some
enzymes to help the reaction
Tightly bound cofactors are called prosthetic groups
Cofactors that are bound and released easily are called
coenzymes
Many vitamins are coenzymes
Types of Cofactors
Coenzyme: The non-protein component, loosely bound to
apoenzyme by non-covalent bond.
Examples : vitamins or compounds derived from vitamins.
Prosthetic group - The non-protein component, tightly
bound to the apoenzyme by covalent bonds.
Some enzymes require cofactors
To take over chemical reactions that cannot be performed by
amino acid side chains
Required in diet of organisms
Organic molecules can associate with enzyme as cosubstrate
(NAD+)
NAD+ obligatory cofactor
In alcohol dehydrogenase reaction
NADH dissociates from the enzyme to be re-oxidized in an
independent reaction
Prosthetic groups
Permanently associated with enzymes
By covalent bonds
Example: heme is bound to proteins called cytochromes
Summary
Enzymes
Transition states
Activation energy
Acid-base catalysis
Covalent catalysis
Metal ion catalysis
Cofactors/coenzymes/cosubstrates/prosthetic groups
The substrate
The substrate of an enzyme are the reactants that are
activated by the enzyme
Enzymes are specific to their substrates
The specificity is determined by the active site
In the lock-and-key model of enzyme action:
-the active site has a rigid shape
-only substrates with the matching shape can fit
-the substrate is a key that fits the lock of the active site
This explains enzyme specificity.
This explains the loss of activity when enzymes denature
This is an older model, however, and does not work for all
enzymes.
Enzymes-how do they work?
Induced-fit hypothesis:
o When a substrate begins to bind to an enzyme, interactions
induce a conformational change in the enzyme
o Results in a change of the enzyme from a low catalytic form
to a high catalytic form
o Induced-fit hypothesis requires a flexible active site
(b)
Catalysis in the Enzyme’s Active Site
In an enzymatic reaction
The substrate binds to the active site
Held by weak interactions (hydrogen bonds)
Side chains (R) from amino acids catalyze the conversion of substrate
to product
Product leaves the active site
Enzyme is free to take another substrate molecule into its active site
Cycle happens very fast
Metabolic reactions are reversible
Enzyme can catalyze both forward and reverse reactions
Enzyme catalyzes the reaction in the direction of equilibrium
Use variety of mechanisms to lower the activation energy and speed
up a reaction
The catalytic cycle of an enzyme
Substrates
Products
Enzyme
Enzyme-substrate
complex
1 Substrates enter active site; enzyme
changes shape so its active site
embraces the substrates (induced fit).
2 Substrates held in
active site by weak
interactions, such as
hydrogen bonds and
ionic bonds.
3 Active site (and R groups of
its amino acids) can lower EA
and speed up a reaction by
• acting as a template for
substrate orientation,
• stressing the substrates
and stabilizing the
transition state,
• providing a favorable
microenvironment,
• participating directly in the
catalytic reaction.
4 Substrates are
Converted into
Products.
5 Products are
Released.
6 Active site
Is available for
two new substrate
molecule
Enzyme Inhibitors
Certain chemicals can inhibit the action of enzymes
Inhibitors
Attach to enzyme by covalent bonds
Usually irreversible
Enzyme Inhibitors Many enzyme inhibitors bind by weak bonds
In that case inhibition is reversible
Some reversible inhibitors resemble the normal substrate molecule
and compete for admission into the active site
Competitive inhibitors
Reduce the productivity of enzymes by blocking substrates from
entering the active site
Competitive inhibitors
Figure 8.19 (b) Competitive inhibition
A competitive
inhibitor mimics the
substrate, competing
for the active site.
Competitive
inhibitor
A substrate can
bind normally to the
active site of an
enzyme.
Substrate
Active site
Enzyme
(a) Normal binding
Noncompetitive inhibitors
Do not directly compete with the substrate
They impede enzymatic reactions by binding to another part of the
enzyme
Causing enzyme to change its shape
Renders the active site less effective at catalyzing the conversion of
substrates to product
Noncompetitive inhibitors
Figure 8.19
A noncompetitive
inhibitor binds to the
enzyme away from
the active site, altering
the conformation of
the enzyme so that its
active site no longer
functions.
Noncompetitive inhibitor
(c) Noncompetitive inhibition
Regulation of enzyme activity helps control metabolism
A cell’s metabolic pathways
Must be tightly regulated
Controlling where and when various enzymes are active
Can be done by switching on and off certain genes that encode specific
enzymes
Allosteric Activation and Inhibition
Many enzymes are allosterically regulated
Have two or more polypeptide chains or subunits
Each has its own active site
The entire complex oscillates between two conformational states:
catalytically active and inactive
Simplest case of allosteric regulation:
Activating or inhibiting regulatory molecule binds to a regulatory site
(located where subunits join)
Binding of activator stabilizes the conformation that has functional
active site
Binding of inhibitor stabilizes inactive form of the enzyme
Subunits fit together so that conformational change in one subunit is
transmitted to all others
Activator or inhibitor that binds to one site will affect the active sites
of all subunits
Stabilized inactive
form
Allosteric activater
stabilizes active from Allosteric enyzme
with four subunits Active site
(one of four)
Regulatory
site (one
of four)
Active form
Activator
Stabilized active form
Allosteric activater
stabilizes active form
Inhibitor Inactive form Non-
functional
active
site
(a) Allosteric activators and inhibitors. In the cell, activators and inhibitors
dissociate when at low concentrations. The enzyme can then oscillate again.
Oscillation
Figure 8.20
Other kind of allosteric activation:
If enzyme has multiple subunits, binding (induced fit) of the
substrate to one subunit can trigger conformational change in all
other subunits
Cooperativity
Amplifies the response of enzyme to substrates
Cooperativity
Figure 8.20
Binding of one substrate molecule to
active site of one subunit locks
all subunits in active conformation.
Substrate
Inactive form Stabilized active form
(b) Cooperativity: another type of allosteric activation. Note that the
inactive form shown on the left oscillates back and forth with the active
form when the active form is not stabilized by substrate.
Feedback Inhibition
In feedback inhibition
The end product of a metabolic pathway shuts down the pathway
Some cells use this pathway to sythesize one amino acid from another
Prevents the cell from wasting chemical resources
Factors affecting enzymes
All enzymes work best at only one particular temperature and pH: this is called the optimum.
Factors that affect the rate of a reaction include:
o substrate concentration
o pH
o enzyme concentration
o surface area
o pressure
o temperature
Different enzymes have different optimum temperatures and pH values.
Factors affecting enzymes
If the temperature and pH changes sufficiently beyond an
enzyme’s optimum, the shape of the enzyme irreversibly
changes.
This affects the shape of the active site and means that the
enzyme will no longer work.
When this happens the enzyme is denatured.
pH and reaction rate
pH also affects the rate of enzyme-substrate complexes
Most enzymes have an optimum pH of around 7 (neutral)
Substrate concentraton and reaction
rate
The rate of reaction increases as substrate concentration
increases (at constant enzyme concentration)
Maximum activity occurs when the enzyme is saturated
(when all enzymes are binding substrate)
Apoenzyme and Holoenzyme
Apoenzyme is the enzyme without its non-protein moiety
and it is inactive.
Holoenzyme is an active enzyme with its non-protein
component.