Hydrolases in Polymer Chemistry: Chemoenzymatic Approaches to Polymeric Materials - Enzymatic Polymerisation - page 96

3.2 Reaction Mechanism and Enantioselectivity of Lipases

Lipases belong to the subclass of -hydrolases and their structure and reaction

mechanism are well understood. All lipases possess an identical catalytic triad con-
sisting of an aspartate or glutamate, a histidine, and a nucleophilic serine residue
[ 67 ]. The reaction mechanism of CALB is briefly discussed as a typical example of
lipase catalysis (Scheme 7 ) .
The catalytic triad of CALB consists of Asp187, His224, and Ser105, while the
oxyanion hole is formed by the backbone amide protons of Thr40 and Gln106 and
the side-chain of Thr40. First, a substrate reversibly complexes to the free enzyme
(Scheme 7 , top left), thereby forming a Michaelis-Menten complex. After correct
positioning of the substrate, a nucleophilic attack of Ser105 onto the substrate car-
bonyl group occurs and a first tetrahedral intermediate is formed (Scheme 7 , top
right). In this tetrahedral intermediate, the negative charge on the former substrate
carbonyl oxygen is stabilized by three hydrogen-bond interactions with the oxyan-
ion hole, whereas the positive charge on His224 is stabilized by interaction with
Asp187. Subsequently, proton transfer from His224 to the substrate alkyl oxygen
takes place and the alcohol part of the residue is liberated from the enzyme. As a
result, a covalently bound acyl-enzyme intermediate is formed at the end of the acy-
lation step (Scheme 7 , bottom right). Subsequently, the acyl-enzyme intermediate
is deacylated by an incoming nucleophile R NuH, which is generally water, an al-
cohol, or an amine. A second tetrahedral intermediate is formed by attack of the
nucleophile onto the acyl-enzyme carbonyl group (Scheme 7 , bottom left). In this
process, the proton is transferred from the nucleophile to the His224 residue and the

α / β

oxyanion
hole

O

Asp187 O

Asp187 O

Ser105 R oxyanion

hole

Ser105

R'

O
H

O R'

O

O -

O -

H H

O -

H

N N

N + O

N

R

His224

His224

Tetrahedral
intermediate 1

Free enzyme

Acylation

O

R''

Nu R'

R OH

Deacylation

Asp187 O oxyanion

hole

Asp187 O

Ser105

Ser105

oxyanion
hole

R'

O

O

O

O -

O -

O -

H

H H

Nu N N

N + N R'

R''

R'' NuH

His224

His224

Tetrahedral
intermediate 2

Acyl-enzyme
intermediate

Scheme 7 Catalytic mechanism of CALB showing an acylation and deacylation step and the
formation of a covalently bound acyl-enzyme intermediate ( bottom right )