Download Lecture 21 Enzyme mechanisms

Lecture 21
Enzyme mechanisms - I
Enzyme binds with the substrate with the help of various interactions like covalent bonding,
hydrogen bonding, ionic interactions, ion-dipole and dipole-dipole interactions, charge
transfer interactions, hydrophobic interactions, and van der Waals interactions. After binding
of the substrate with the active site, the enzyme converts the substrate into the product by
employing various types of catalytic mechanisms. These mechanisms are classified as:
1. Acid–base catalysis.
2. Covalent catalysis.
3. Metal ion catalysis.
4. Electrostatic catalysis.
5. Proximity and orientation effects.
6. Preferential binding of the transition state complex.
1. Acid–Base Catalysis
In general acid catalysis process, lowering of free energy of the transition state occurs via
partial proton transfer from a Brønsted acid. For example, keto–enol tautomerization reaction
occurs quite slowly when it is uncatalysed. The carabanionlike transition state of this
reaction, being high energy slows down the reaction rate. In the presence of acid, donation of
proton to the oxygen atom reduces carbanion character of the transition state and hence
increases the reaction rate. Few reactions also may be catalyzed by a Brønsted base. Also
there are some reactions where simultaneous catalysis by acid and base occur.
The RNase A Reaction Incorporates General Acid–Base Catalysis
An example of enzymatic acid–base catalysis is Hydrolysis of RNA to its component
nucleotides by Bovine pancreatic ribonuclease A (RNase A). This digestive enzyme cleaves
RNA using the following reaction sequence:
Mechanism
of RNA
(Image
hasCleaving:
to be drawn later)
Active site of RNase A comprises of two His residues (His 12 and His 119) and Lys 41
which act in a concerted manner as general acid and base catalysts. RNase A cleaves RNA
by following the two-step process:
1. In the first step, His 12 behaves as a general base. It abstracts a proton from 2ʹ-OH group
of RNA. The newly generated 2ʹ-O-, being a good nucleophile promptly attack on the
adjacent phosphorous atom. In the concerted way His 119 acts as an acid by protonating the
oxygen atom of the leaving group which promotes the bond scission between phosphorous
and 5ʹ oxygen. As a result of this step a 2ʹ,3ʹ-cyclic intermediate is formed.
2. In the second step, actually reverse of the first step take place. Here, His 12 acts as a
general acid and His 119 as a general base. His 119 abstract a proton from a water molecule
and facilitate the nucleophilic attack to the phosphorous atom of the 2ʹ,3ʹ-cyclic intermediate.
His 12 protonate the 2ʹ-oxygen atom of the cyclic intermediate and helps to cleave the bond
between phosphorous and 2ʹ-oxygen atom.
2. Covalent Catalysis
In the covalent catalysis process a transient catalyst–substrate covalent bond is formed. An
example of such a process is decarboxylation of acetoacetate which is chemically catalyzed
by primary amines.
In the uncatalyzed decarboxylation process the enolate transition state is very unstable.
Whereas in the catalytic process, the high-energy enolate character of the transition state is
stabilized as the protonated nitrogen atom of the covalent intermediate acts as an electron
sink. The steps of Schiff base formation as well as its decomposition are very fast, so that
these steps are not rate determining in this reaction sequence.
Covalent catalysis is the summation of three successive three stages:
1. The nucleophilic reaction between the catalyst and the substrate to form a covalent bond.
2. The withdrawal of electrons from the reaction center by the now electrophilic catalyst.
3. The elimination of the catalyst, a reaction that is essentially the reverse of stage 1.
D. Electrostatic Catalysis
When substrate binds with the enzyme it generally excludes water from the active site. Due
to the presence of substrate at the active site, the local dielectric constant of the active site is
similar to that as found in an organic solvent. As a result the electrostatic interactions are
much stronger than they are in aqueous solutions. Here, the distribution of charge is present
in a medium of low dielectric constant which greatly influences chemical reactivity. Due to
the presence of charge group near the active site, the pK’s of amino acid side chains in
proteins may vary by several units from their nominal values. Distributions of charge near
the active sites of enzymes are arranged in such a way that it can stabilize the transition states
of the catalyzed reactions which in turn increase the rate of the reaction. Enhancement of
enzymatic reaction rate due to the stabilization of transition state by the charge distribution is
termed electrostatic catalysis. In case of few enzymatic reactions these charge distributions
acts as a guide of a polar substrates to reach toward their binding sites. As a result rate of
these reactions is higher than that of their apparent diffusion-controlled limits.
Catalysis through Proximity and Orientation Effects
Catalytic mechanisms of enzymatic reaction are similar to that of organic model reactions. In
spite of this, enzymatic reactions are far more
catalytically efficient than these models. Higher
efficiency of enzymatic reaction is due to the
specific physical conditions at enzyme catalytic
sites that promote the corresponding chemical
reactions. Proximity and proper orientation of the
substrate and active site of an enzyme are the two
most obvious specific physical conditions. For a
reaction to occur, reactants must come together
with the proper spatial relationship.
For example, bimolecular reaction of imidazole
with p-nitrophenylacetate.
The progress of the reaction is monitored by the
formation of the intensely yellow p-nitrophenolate
ion.
But if the reaction proceeds intramolecularly, the first-order rate constant k2 = 24 k1, when
concentration of imidazole is 1M. Thus, due to the attachment of the imidazole catalyst with
the the reactant, it is 24-fold more effective
than when it is free in solution. So, in the case
intramolecular reaction the imidazole group
behaves as if its concentration is 24M. Both
proximity and orientation contribute to this rate enhancement.
Catalysis by Preferential Transition State Binding
The catalytic mechanisms so far discussed for an enzyme is not the enough reason for the
enormous rate enhancements effected by enzymes. One of the most important mechanisms of
enzymatic catalysis is not considered yet i.e., the binding of the transition state to an enzyme
with greater affinity than the corresponding substrates or products. If both of them i.e. the
previously described catalytic mechanisms and preferential transition state binding are
consider together that will rationalizes the observed rates of enzymatic reactions. The
concept of transition state binding is based on rack mechanism. The concept proposed that
enzymes mechanically strained their substrates toward the transition state geometry through
binding sites into which undistorted substrates did not properly fit.
Model (organic reaction) example:
The reaction occurs at 315 times faster rate when R is CH3 rather than when it is H due to the
greater steric repulsions between the CH3 groups and the reacting groups. Another example
of reaction rate enhancement due to strain is the ring opening reaction where a strained ring
such as cyclopropane opens at faster rate compared to the unstained ring such as
cyclohexane. In case of both of the mentioned example the strained reactant is more close to
the transition state of the reaction in terms of energy than does the corresponding unstrained
reactant.