Download A group on the enzyme acts as an acid or base

Lecture 12:
Enzyme Catalysis
Catalytic Strategies
Steps in a Reaction
Enzymes are Classified According to the Reactions They Catalyse
But while the details of the particular reaction vary from
enzyme to enzyme, similar strategies are used to carry them out.
Common Types of Catalysis
Covalent Catalysis:
A group on the enzyme becomes covalently modified during
reaction, e.g. by forming a covalent bond to the substrate
during the reaction.
General Acid-Base Catalysis:
A group on the enzyme acts as an acid or base: it removes a
proton from or donates a proton to the substrate during
the reaction.
Metal Ion Catalysis:
A metal ion is used by the enzyme to facilitate a chemical
rearrangement or binding step.
Catalysis by Approximation:
The enzyme holds two substrates near in space and in precisely
the correct spatial orientation to optimize their reaction.
(Most enzymes use a combination of several of these strategies)
Covalent Catalysis
Rate acceleration by transient formation of a
COVALENT enzyme-substrate bond
Enzyme
Substrate
Covalent
Intermediate
Enzyme
Products
Reaction can’t go back because displaced group has been released.
The enzyme alters pathway to get to product by stabilizing the
intermediate with a covalent bond.
But: if a covalent bond is formed at an intermediate step, the bond
must be broken in a subsequent step to finish the reaction.
Covalent Catalysis Requires a Highly Reactive Group
Some chemical group that can invade the substrate- usually a nucleophile.
Nucleophile: an electron-rich group that attacks nuclei.
Examples of nucleophiles among protein functional groups:
unprotonated His imidazole
unprotonated a-amino group
unprotonated sidechain amino group of Lys
thiolate anion (-S-) of Cys
aliphatic -OH of Ser
sidechain carboxylates of Glu, Asp
(Also some coenzymes.)
General Acid-Base Catalysis
Specific functional groups in enzyme structure are positioned to
either
Donate a proton (act as a general acid)
or
Accept a proton (act as a general base).
This enables enzyme to avoid unstable charged intermediates in reaction,
so as to keep the transition state in a stable (low-energy) state
But: A group that donates a proton (acts as a general acid) in catalysis has to
then accept a proton (act as a general base) later in catalytic mechanism
for catalyst to be regenerated in original form.
Examples of general acid/base catalysts among protein functional groups:
His imidazole
a-amino group
thiol of Cys
R group carboxyls of Glu, Asp
Sidechain amino group of Lys
Aromatic OH of Tyr
Guanidino group of Arg
Metal Ion Catalysis
Metal ions can be used in a variety of ways by enzymes. (In fact they are so
useful that about one-third of enzymes use them for one thing or another.)
Binding and orientation of substrate:
By forming strong ionic interactions with substrate, it can be
precisely oriented. (Especially strong because when water is
excluded from active site, the dielectric constant is quite low.)
Redox reactions:
Ions that have more than one possible charge
state (eg. iron Fe2+ and Fe3+) can gain or lose electrons during
the reaction, avoiding unstable charged intermediates.
Shielding or stabilizing negative charges:
If charge on substrate or on transition state is an integral
part of the reaction, the enzyme can form strong ionic interactions
with that charge which are stabilizing.
Enzyme
Substrate
+2
Binding and orientation of substrate
AND/OR
Shielding or stabilizing negative charges
Unstable
+2
+
Stable
+3
0
Redox
Reactions
Catalysis by Approximation
Proximity: Reaction between bound molecules doesn't require an improbable
collision of 2 molecules. They're already in "contact" (increases local
concentration of reactants).
Orientation: Reactants are not only near each other on enzyme, they're
oriented in optimal position to react. The improbability of colliding
in correct orientation is taken care of.
Substrates held close in space
and in correct orientation
Results in more
efficient reaction pathway
Other Effects that Stabilize the Transition State
Electrostatic Effects:
Increase in strength of ionic interactions due to lower
dielectric constant.
Desolvation:
Exclusion of water from active site.
Induced Fit:
Change in conformation of enzyme or substrate to
optimize interactions.
Electrostatic Effects
Enhancement of the attraction between opposite charges
by various means.
Coulomb’s Law:
q1q2
F k
2
Dr
Examples:
Providing a lower dielectric constant of the environment in the
active site (hydrophobic environment)
Altering pK values of specific functional groups.
Stabilizing a particular conformation of the critical groups in
the active site by electrostatic interactions.
Stabilizing (binding) a charged intermediate or transition state by
providing an oppositely charged enzyme group close by.
Desolvation
By sequestering reactants away from water, two major effects are
Achieved:
Lower dielectric constant environment than in water.
results in stronger electrostatic interactions.
Reactive groups of reactants are protected from H2O,
so H2O doesn't compete with reactants or affect equilibrium.
Induced Fit
Conformational change resulting from substrate binding
may stabilize a different conformation of either enzyme
or substrate or both.
Conformational change can:
Promote faster chemical steps
(eg by orienting catalytic groups on enzyme
Promote tighter transition state binding
(eg by orienting binding groups on enzyme)
Close off active site and sequester reactants away from water.
Examples:
Chymotrypsin- hydrolysis of peptide backbone
Incorporates covalent catalysis and acid-base catalysis
(details next lecture)
Carbonic Anhydrase- equilibrate carbon dioxide and carbonic acid
Metal ion catalysis
(near catalytic perfection)
Restriction Enzymes- sequence-specific cleavage of DNA backbone
hydrolysis via covalent catalysis
(extreme specificity)
NMP Kinases- transfer of phosphate group between 2 substrates
group transfer using metal ion catalysis
(very efficient- avoid loss of the phosphate group)
Reactions Proceed in a Series of Steps
S
Net Reaction:
P
Enzyme Catalysed Pathway:
Chemical
Rearrangement
Substrate
Binding
E + S
ES
EP
Product
Release
E + P
Each of these steps might be analysed further to understand atomic details.
Group Transfer Reactions Have 2 Substrates
S1-G + S2
Net Reaction:
A+B
2 Substrates
S1’ + S2’-G
P+Q
2 Products
Substrates and Products can be bound and released in various orders:
Sequential Displacement:
Ordered Sequential
Random Sequential
Double Displacement
Ordered Sequential Reactions
(e.g. lactate dehydrogenase)
All substrates bind before any product is released.
(protons tranferred)
Substrate
Binding
Steps
Chemical
Rearrangement
Steps
Product
Release
Steps
Random Sequential Reaction
(e.g. creatine kinase)
Order of substrate binding and product release is random.
(phospate
tranferred)
Substrate
Binding
Steps
Chemical
Rearrangement
Steps
Product
Release
Steps
Double Displacement (or Ping-Pong) Reaction
(e.g. aspartate aminotransferase)
Some products released before all substrates are bound.
(amino group
tranferred)
Substrate
Product
Binding
Release
Step
Step
Chemical
Rearrangement
Step
(amino group
on enzyme)
Substrate
Product
Binding
Release
Step
Step
Chemical
Rearrangement
Step
Summary:
The chemical reactions catalyzed by most enzymes can be classified
into one of 6 general types of reactions.
A few catalytic strategies are used by most enzymes regardless of
the particular chemistry they perform.
Enzyme-catalyzed reactions proceed in an organized series of steps
each of which can be considered separately.
Key Concepts:
Covalent catalysis
Nucleophile
Acid-Base Catalysis
Metal-ion Catalysis
Catalysis by Approximation
Electrostatic Effects, Desolvation, Induced Fit
Displacement reactions