Download Enzymology

Enzymatic 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 (eacceptor)
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?
•
•
•
•
•
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
at low pH Enz - -> EnzH
at 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:
Remember allosteric regulations?
• Allosteric interaction – of, relating to,
undergoing, or being a change in the shape and
activity of a protein (as an enzyme) that results from
combination with another substance at a point other
than the chemically active site
• a regulatory mechanism where a small
molecule (effector) binds and alters
an enzymes activity
Allosteric interaction
• Each
allosteric
unit,
called
a protomer (generally assumed to be a
subunit), can exist in two different
conformational states - designated 'R'
(for relaxed) or 'T' (for tense) states.
• The R state has a higher affinity for
the ligand than the T state.
• Because of that, although the ligand
may bind to the subunit when it is in
either state, the binding of a ligand will
increase the equilibrium in favor of the
R state.
Allosteric interaction
• Allosteric sites are sites on the enzyme
that bind to molecules in the cellular
environment.
• The sites form weak, noncovalent bonds
with these molecules, causing a change
in the conformation of the enzyme.
• This change in conformation translates
to the active site, which then affects
the reaction rate of the enzyme.
•
Allosteric interactions can both inhibit
and activate enzymes and are a common
way that enzymes are controlled in the
body.
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 (pp 323)
 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.
•
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.