Download Lecture 22 Enzyme mechanisms - II

Lecture 22
Enzyme mechanisms - II
Covalent catalysis are arbitrarily classified as nucleophilic catalysis or electrophilic
catalysis. The mechanism of nucleophilic catalysis and general base catalysis are quite
similar except that, instead of abstracting a proton from the substrate, the catalyst
nucleophilically attacks it so as to form a covalent bond. In covalent catalysis if the covalent
bond between catalyst and substrate is more stable the formed, then it will decompose less
facilely in the final steps of a reaction. So a good covalent catalyst is one which is a good
nucleophile as well as a good leaving group.
Example of Covalent catalysis:
(1) Hydrolysis of β(1→4) Linkages from N-acetylmuramic acid (NAM) to Nacetylglucosamine (NAG) by Lysozyme:
Lysozyme is an enzyme that destroy bacterial cell wall by hydrolyzing the β(1 → 4)
glycosidic linkages from N-acetylmuramic acid (NAM) to N-acetylglucosamine (NAG) in
the alternating NAM–NAG polysaccharide component of cell wall peptidoglycans. It
likewise hydrolyzes β(1 → 4)-linked poly(NAG) (chitin), a cell wall component of most
fungi.
The mechanism of hydrolysis is as follows:
1. Lysozyme attaches to a bacterial cell wall by binding to a hexasaccharide unit. In the
process, the D-ring is distorted toward the half-chair conformation in response to the
unfavorable contacts that its −C6H2OH group would otherwise make with the protein.
2. Glu 35 transfers its proton to the O1 atom linking the D- and E-rings, the only polar group
in its vicinity, thereby cleaving the C1−O1 bond (general acid catalysis).This step converts
the D-ring to a planar resonance-stabilized oxonium ion transition state, whose formation is
facilitated by the strain distorting it to the half-chair conformation (catalysis by the
preferential binding of the transition state).The positively charged oxonium ion is stabilized
by the presence of the nearby negatively charged Asp 52 carboxylate group (electrostatic
catalysis).The E-ring product is released.
3. The Asp 52 carboxylate group nucleophilically attacks the now electron-poor C1 of the D
ring to form a covalent glycosyl–enzyme intermediate (covalent catalysis).
4. Water replaces the E-ring product in the active site.
5. Hydrolysis of the covalent bond with the assistance of Glu 35 (general base catalysis),
which involves another oxonium ion transition state, regenerates the active site groups. The
enzyme then releases the D-ring product, completing the catalytic cycle.
Example 2. SERINE PROTEASES
Another example of covalent catalysis is a diverse group of proteolytic enzymes known as
the serine proteases. Their name ‘serine proteases’ comes from the presence of a reactive
Ser residue in their active site and this residue takes part in catalytic activity following a
common mechanism. We will discuss the mechanism of chymotrypsin (a serine protease)
mediated proteolysis.
1. First chymotrypsin binds to the substrate to form the Michaelis complex. Then in the ratedetermining step of the reaction, Ser 195 nucleophilically attacks to the carbonyl group of
scissile peptide to form a complex known as the tetrahedral intermediate (covalent catalysis).
His 57 abstract the liberated proton to form an imidazolium ion (general base catalysis). Asp
102 is hydrogen bonded to His 57. The polarizing effect of the unsolvated carboxylate ion of
Asp 102 helps His 57 to abstract the proton.
2. In the second step, N3 of His 57 donates a proton to the N atom of tetrahedral
intermediate. It provides driving force for the decomposition of tetrahedral intermediate into
the acyl–enzyme intermediate. The leaving group (RʹNH2) of the reaction is the new Nterminal portion of the cleaved polypeptide chain. It is released from the enzyme and
replaced by water from the solvent.
3 & 4. In the third step, deacylation of the acyl–enzyme intermediate takes place rapidly.
Water nucleophilically attacks to the acyl carbon of the acyl–enzyme intermediate which is
facilitating by the abstraction of proton by His 57 from that water molecule. The process is
followed by the leaving of Ser 195 which in turn results carboxylate product (the new Cterminal portion of the cleaved polypeptide chain).
3. Metal Ion Catalysis
Presence of metal ions is quite essential for many enzymes to show catalytic activity.
Depending on the strengths of their ion–protein interactions metal ion catalyst can be
classified into two subclasses:
a. Metalloenzymes: Here, the protein part of the enzyme is tightly bound with metal ions.
These metal ions are generally transition metal ion, such as Fe2+, Fe3+, Cu2+, Zn2+, Mn2+, or
Co3+.
b. Metal-activated enzymes: Here, the protein part of the enzyme is loosely bound with
metal ions from solution. These metal ions are usually the alkali and alkaline earth metal
ions, such as Na+, K+, Mg2+, or Ca2+.
There are three major ways by which metal ions participate in the catalytic process in:
I. By binding to substrates so as to orient them properly for reaction.
II. By mediating oxidation–reduction reactions through reversible changes in the metal ion’s
oxidation state.
III. By electrostatically stabilizing or shielding negative charges.
Among the three major ways, the third aspect of metal ion catalysis will be discussed here.
a. Metal Ions Promote Catalysis through Charge Stabilization
Similar to proton, metal ion acts by neutralizing negative
charge in many metal ion–catalyzed reactions. Here, the metal
ions behave as a Lewis acid. As a catalyst Metal ion has
advantage over proton as metal ions can be present in high
concentrations at neutral pH’s can have charges greater than
+1. So, Metal ions are more effective catalysts compared to
protons
An example nonenzymatic catalysis by a metal ion is the
decarboxylation of dimethyloxaloacetate catalyzed by metal
ions such as Cu2+ and Ni2+.
b. Metal Ions Promote Nucleophilic Catalysis via Water Ionization
When a metal ion binds with water molecules, the charge of the metal ion causes that water
molecule to be more acidic than free H2O and therefore a source of OH− ions even below
neutral pH’s. For example, the water molecule of (NH3)5Co3+ (H2O) ionizes according to the
reaction:
(NH3)5Co3+ (H2O) ↔ (NH3)5Co3+ (OH−) + H+
Carbonic anhydrase:
An example of mechanism of metal ion catalysis is catalytic mechanism of the following
reaction by carbonic anhydrase:
CO2 + H2O ↔ HCO3− + H+
In carbonic anhydrase a Zn2+ ion is
present at the active site cleft. The
central metal atom is tetrahedrally
coordinated by three imidazole (Im)
ring of three His side chains and an
O atom of either an HCO3− ion or a
water molecule. The enzyme acts by
the sequence of following catalytic mechanism:
1. At first, the metal ion i.e. Zn2+ polarizes H2O molecule. This water molecule then ionizes
in a process facilitated through general base catalysis by His 64. Although His 64 is too far
away from the Zn2+-bound water to directly abstract its proton, these entities are linked by
two intervening water molecules to form a hydrogen bonded network that is thought to act as
a proton shuttle.
2. The OH− ion resulting from the former
step, nucleophilically attacks the nearby
enzymatically bound CO2, thereby
converting it to HCO3−. The optimal
geometry of OH− for nucleophilic attack
on the substrate CO2 is attaining by the
existence of two hydrogen bond. The first
hydrogen bond is between Zn2+-bound
OH− group with Thr 199 and the second
one is between Thr with Glu 106
3. In the final step, the catalytic site is regenerated. Here, water molecule completely replaces
HCO3− from the Zn2+-bound HCO3− and deprotonation of His 64 also occur.