Download Acid-Base Catalysis

Chapter 11
Enzymatic catalysis
Role of enzymes
 serve the same role as any other catalyst in chemistry
 act with a higher specificity
 acid and base catalyst possible due to proximal locations of
amino acids
 alter the structure location of quantity of the enzyme and you
have regulated the formation of product - try that in organic
chemistry !
 Don't lose sight of what enzymes do. Straight forward
reactions…. reactants -> products.
Naming enzymes  Enzyme commission for each enzyme based on the type of
reactions. Kind of like IUPAC
 Yeah right - the mother of invention – he/she who finds it
names if. Trivial names rule
Enzyme reaction types: Enzymes are classified
into six major classes based on the reaction
they catalyze.
1) Oxidoreductases catalyze oxidation-reduction
reactions. Most of these enzymes are known as
dehydrogenases, but some are called oxidases,
peroxidases, oxygenases or reductases.
A- + B -> A + BA is the reductant (e- donar) and B is the oxidant (e- acceptor)
NAD+ (ox) +e- + H+ -> NADH (red)
catalyzes the transfer of electrons from one molecule (the reductant, also
called the hydrogen or electron donor) to another (the oxidant, also called the
hydrogen or electron acceptor).
2) Transferases catalyze group-transfer reactions
(functional groups). Many require the presence of
coenzymes. A portion of the substrate molecule
usually binds covalently to these enzymes or their
coenzymes. This group includes my personal favorite,
the kinases
A-X + B -> A + B-X
A is the donar and B is the acceptor Donor s often a coenzyme
ATP + R-OH -> ADP + R-PO4-2
3) Hydrolases catalyze hydrolysis. They are a special
class of transferases, with water serving as the
acceptor of the group transferred
A-B + H2O -> A-OH + B-H
ie Glycoside hydrolases (also called glycosidases) catalyze the
hydrolysis of the glycosidic linkage to generate two smaller
sugars.
They are extremely common enzymes with roles in nature including
degradation of biomass such as cellulose and hemicellulose
Together with glycosyltransferases, glycosidases form the major
catalytic machinery for the synthesis and breakage of glycosidic
bonds.
4) Lyases catalyze nonhydrolytic and nonoxidative
elimination reactions, or lysis, of a substrate,
generating a double bond. In other words these
reactions catalyze group elimination to form double
bonds. In the reverse direction, lyases catalyze
addition of one substrate to a double bond of a
second substrate.
A lyase that catalyzes addition reaction in cells is often
termed a synthase.
ATP -> cAMP + PPi
Usually only need one substrate in one direction and two
for the reverse!
5) Isomerases catalyze the isomerase reactions.
catalyses the structural rearrangement of isomers
A -> B
Because these reactions have only one substrate and
one product, they are among the simplest enzymatic
reactions.
Names of these enzymes are “substrate isomersase”
Ex. - enoyl CoA isomerase
catalyzes conversion of cis-double bonds of fatty acids at position
3 to trans double bonds at position 2. It has a special importance
in metabolism of unsaturated fatty acids
or some sort of mutase a to b form of carbohydrate
6) Ligases catalyze ligation or joining, of two substrates.
These reactions require the input of chemical potential
energy of a nucleoside triphosphate such as ATP. i.e.
bond formation coupled to ATP hydrolysis. Ligases are
usually referred to as synthetases.
Ab + C -> A-C + b
Sometimes involve a hydrolysis
A and C are both large polymers DNA ligase
General Information on Enzymology
Enzyme nomenclature
• Active site
• Substrate vs. reactant
• Prosthetic groups, coenzyme and cofactors
• Activity vs. reaction
• Suffix -ase
Another important reminder - not all activity is on or off. Many
times the enzyme has a low or constitutive activity that can
be increased many times. It is a common mistake to think
of enzymes being on or off.
Enzyme specificity
- One of the most powerful actions of enzymes.
– Group complementation - the ability to recognize specific
regions of the substrate to align reactants with catalytic
site.
– Based on non-covalent molecular interactions.
– Lock and key vs. induced fit - both occur. Induced fit
takes place when binding of one part of the substrate to
the enzyme alters the conformation of the enzyme to
make a true "fit"
– Binding does not mean activity
Enzyme specificity
An enzyme can bind and react stereo-specifically with chiral
compounds
• This can happen due to a three point attachment
– Binding can then only occur in one way and therefore
the products are not a mixture.
Substrate
Enzyme binding site
3 pt attachment demonstrating the sterio-specificity
on an enzyme binding to its substrate
How do enzymes work?
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By reducing the energy of activation
This happens because the transition state is stabilized by a number of
mechanisms involving the enzyme/protein
Without enzymes reactions occur by collisions between reactants or addition of
various organic catalysts
The energy barrier between the reactants and product, is the free energy of
activation.
The free energy (G) of
the reaction is what
determines if a reaction
is spontaneous.
How do enzymes work?
• The rate is determined by That's why an enzyme can increase the speed
/ rate of a reaction.
• The overall free energy of a reaction, G, is independent of the free
energy of activation. So if the G is less than zero the reaction will
proceed once a catalyst is added (the enzyme).
– But the converse is also true - the reverse reaction is also able to
proceed depending on the G. Some reactions have very little
changes free energy
and are freely
reversible. Availability
of enzyme (catalyst)
and the relative
concentrations of
products and
reactants will decide
which direction these
reactions will go.
Factors effecting catalysis
Temperature – Unlike conventional chemical reactions,
increasing temperature of a reaction can have mixed
results.
 Initial increases in temperature result in higher rates of
reaction due to increasing the kinetic activity of reacting
molecule – more forceful collisions and higher rates of
diffusion between substrate and its enzyme
 Increased temp exceeds energy barrier to maintain
hydrogen and hydrophobic bonds of the enzyme. This
results in denaturation of and loss of enzyme function
Factors effecting catalysis
pH – There is typically an optimum pH of a reaction for both
substrate and enzyme. The aa in the catalytic site as well
as for structure is vital for the reaction to occur. Most
enzymes are stable in the pH range of 5 – 9.
Example - Enz- + SH+ -> EnzSH
@ low pH Enz - -> EnzH
@ high pH SH -> S- + H+
General catalytic mechanisms of enzyme
reactions
- also known as the exact means that enzymes lower the transition
state and catalyze the reaction.
• There are six general mechanisms by which enzymes act. These
are the contributing factors of enzyme catalysis:
Acid-Base Catalysis Chemical groups can often be made more reactive
by adding or removing a proton.
Enzyme active sites contain side chain groups that act as proton
donors or acceptors.
These groups are referred to as general acids or general bases. The
amino acid used in the reaction depends on the pH such that the
side chain is in the appropriate charge state, and the local
environment.
Histadine is a good example of this. The side chain of
histidine has a pKa of around 6. Therefore the side chain can
undergo ionization at a physiological pH.
The protonated form is a general acid and ionized form is the general
base. The task of a catalyst is often to make a reactive group more
reactive by increasing its intrinsic electrophilic or neutrophilic
character.
The easiest way to do this is to remove or add a proton. It is not
practical to undergo this mechanism by free acids or bases and at
physiological pH, the concentration of OH- and H+ is too low to
induce the catalysis.
Therefore the correct placement of an amino acid that can act as a
base or acid effectively increases the concentration at the correct
location of the substrate.
 See box 11-1 for pH effects on catalysis
 RNase A is an example of this type of reaction
Covalent Catalysis In some enzymes a nucleophilic side
chain group forms an unstable covalent bond with
the substrate. The enzyme-substrate complex them
forms product. The pathway can require that the
intermediate is more susceptible to nucleophilic
attack by water than the original substrate.
• Three stages of covalent catalysis:
 Formation of a bond between substrate and
enzyme
 Removal of electrons to make a reactive center
 Elimination of the bond that was formed in step
one
Metal Ion Catalysis Transition metals as well as divalent
cations (Mg+2 and Ca+2) are useful for this type of
catalysis. Metal ions act as a lewis acid and accept
electrons. Therefore they are effective electrophiles.
Another important reason for involving metals is the
positive charge at any physiological pH.
 Involved in redox reactions
 Metals such as zinc activate water - to "acidify" or
polarize the water so the OH group can act as a
nucleophile.
 Carbonic anhydrase is an example of this type of
reaction
Zn2+ in carbonic anhydrase – Zn2+ is coordinated by 3 imidazole groups
and a water molecule.
(Arrow shows opening of active site)
Proximity and Strain (Orientation) Effects For a biochemical
reaction to occur, the substrate most come into close proximity
to catalytic functional groups (side chains of amino acids which
are involved in catalytic reactions) with in the active site. In
addition, the substrate must be precisely oriented to the
catalytic groups. Once the substrate is correctly positioned, a
change in the enzyme's conformation may result in a strained
enzyme substrate complex.
This strain helps to bring the
enzyme substrate complex into the transition state.
In general, the more tightly the active site can bind the substrate
while it is in the transition state, the greater the rate of the
reaction
Proximity alone can only account for a five fold increase in
activity
Without the proper orientation, little reaction can occur the Sn2 attack is a good example
Lack of orientation is the key for an increased rate of
reaction
Electrostatic Effects Recall that the strength of electrostatic
interactions is related to the capacity of surrounding solvent
molecules to reduce the attractive forces between chemical
groups. Because water is largely excluded from the active
site of most enzymes, the local dielectric constant is low. The
charge distribution in the relatively anhydrous active site may
influence the chemical reactivity of the substrate.
In addition, weak electrostatic interactions such as those
between permanent and induced dipoles in both the reactive
site and the substrate are believed to contribute to catalysis.
A more efficient binding of substrate lowers the free energy of
the transition state, which accelerates the reaction. Thus
enzymes act by stabilizing the distribution of electrical charge
in the transition states.
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Many inhibitors of enzymes mimic the transition state. The enzyme binds
the inhibitor at a stable energy state - these inhibitors bind with a much
higher affinity than the normal substrate.