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3 Candida antarctica Lipase B–Characterization of a Versatile Biocatalyst

3 Candida antarctica Lipase B–Characterization of a Versatile Biocatalyst

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354



14 Molecular Modeling Approach to Enzymatic Polymerization



serine. This preferential binding is responsible for much of the catalytic efficiency

and the stereoselectivity of the enzyme and it is of utmost importance for the

enzymatic polymerization as described later on in this article.

Besides the crystal structure of the native enzyme, the PDB database [3] provides two further CALB crystal structures elucidated by Uppenberg et al. [6],

which characterize complexes of the enzyme with small ligands. Of these structures the PDB code 1LBT describes the complex with a molecule of the detergent

Tween 80 embedded in the active site while the PDB code 1LBS stands for the

crystal structure of phosphonate inhibitor covalently bound to a Ser105 (Figure

14.3b). These structures can be used as a starting point for molecular modeling

investigations.



14.4

Lipase Catalyzed Alcoholysis and Aminolysis of Esters



Lipases are usually active in aqueous systems as well as in organic solvents which

makes them applicable not only for hydrolytic reactions but also for esterifications

and transesterifications or for the formation of amides. These enzymes are known

to accept a wide variety of substrates quite often with high enantioselectivity which

makes them useful for chiral organic substrates. Numerous molecular modeling

studies during the last decade describe these phenomena and provide us today

with a clear picture of the mechanisms behind the enzymatic reaction particularly

of CALB. Vicente Gotor, Karl Hult, and Romas J. Kazlauskas are three of the most

cited names in this regard who have contributed fantastic work in this field.

First attempts to predict the selectivity of enzymes are dated back to 1964 when

Prelog described an empirically determined rule for the addition of hydrogen to

ketones by the yeast Culvaria lunata [13]. In 1991 Kazlauskas published the

hydrolysis of acetates of secondary alcohols by Pancreatic cholesterol esterase,

Pseudomonas cepacia and Candida rugosa and formulated the widely applicable

Kazlauskas rule according to which esters of secondary alcohols with a specific

substitution pattern of large (L) and medium (M) substituents are cleaved faster

than the corresponding enantiomer [14].



However, due to the diversity of the active sites in different enzymes there

is no universally valid rule for all hydrolases but rather individual enzymes



14.4 Lipase Catalyzed Alcoholysis and Aminolysis of Esters



(a)



Figure 14.4 Tetrahedral intermediates of



CALB aziridine - carboxylate complexes. The

slow reacting (2R,1’R)- diastereomer (b)

exhibits an umbrella-like inversion of

substituents compared with the fast reacting



(b)



(2S,1’R)- diastereomer (a). The hydrogen at

the 2R stereocenter interferes with the

hydroxyl group of Thr40 (orange), and one of

the three hydrogen bonds of the oxyanion

hole is lost (blue line) [11a].



control stereospecific ester or amide cleavage on a specific structural basis.

A recent example from the Kazlauskas group for instance explains the enantiopreference of CALB towards the ammoniolysis of aziridine-2- carboxylates [11a]

which is an extension of the classical Kazlauskas rule (Figure 14.4). While in

this example the fast reacting (2S, 1’R)- diastereomer of the serine bound ligand

perfectly fits to the active site for aminolysis (Figure 14.4a), the slow reacting

(2R, 1’R)- diastereomer, can only form the tetrahedral intermediate at all after

an umbrella like inversion of the substituents at the ring-stereocenter. This

results in an unfavorable interaction with Thr40 and in the loss of one hydrogen

bond of the oxyanion hole which in turn is responsible for the reduced reaction

rate.

A rather different sterospecificity has recently been described by the Hult group

for the esterification of pentan-2- ol with propionic acid methylester [11d]. It is well

known that the water content in the enzyme – and in particular in the binding

site – has a pronounced effect on both reaction rate and enantioselectivity. The

presence of too much of water would prevent transesterification, while too low a

water content destabilizes the enzyme, resulting in decreased reactivity. In this

specific example the enantioselectivity is explained by the influence of a thermodynamic water molecule on CALB (Figure 14.5). While in the (R)-intermediate

the propyl group of the chiral center is oriented towards the entrance of the

pocket (Figure 14.5a) in the (S )-intermediate it points directly towards the stereospecificity pocket (Thr42 and Ser47) that contains the decisive water molecule



355



356



(a)



14 Molecular Modeling Approach to Enzymatic Polymerization

(b)



Figure 14.5 Models of (a) the (R)- and

(b) the (S )-tetrahedral intermediates

of 2-pentylpropanoic acid with a

water molecule introduced into the

stereospecificity pocket (Thr24 andSer47).

The water molecule inside this



pocket forms five hydrogen bonds in

presence of the (R)- enantiomer while

only three hydrogen bonds can be formed

in the case of the (S )- enantiomer which

thermodynamically favors the (R)enantiomer [11d].



(Figure 14.5b) thereby reducing the number of hydrogen bonding of this water

from five in the (R)- enantiomer to only three in the (S )- enantiomer, which indicates a better fit for the water molecule in the (R)- enantiomer and an unfavorable

enzyme conformation necessary for the (S )- enantiomer to react.

Finally, a third example should serve further to demonstrate the diversity of

stereoselective reactions provided by CALB. In a recent publication the Gotor

group suggests the formation of zwitterionic species, resulting from the direct

His-unassisted attack of an amine onto the carbonyl group of the acyl– enzyme,

as the most plausible intermediate for the CALB - catalyzed aminolysis [11f]. This

proposal differs slightly from the commonly accepted serine-mediated mechanism, where removal of the proton from the amine occurs simultaneously with

the nucleophile attack to the acyl– enzyme complex. Subsequently, His-assisted

deprotonation of the resulting ammonium group takes place, and a molecule of

water in some cases is necessary to facilitate the transfer of the proton to the catalytic histidine. Proton transfer-aided pathways have been previously proposed for

CALB - catalyzed reactions in which modeling predicted that a molecule of water

is necessary to transfer the proton from the nucleophile to the catalytic histidine

[10c, 15]. Stereoselectivity is achieved in this example by better binding of the

substrate, together with a molecule of water in the (1R,2S)-2- cyclohexanamide

intermediate, justifying the enantiopreference exhibited by CALB towards this

substrate (Figure 14.6).



14.6 CALB -Catalyzed Polymerization of β-Lactam



Me



Ser105

O



His224

Asp187



COO



H N



N



H O

H



P O

H NH

(R) Ph

(S)



oxyanion

hole



Figure 14.6 Geometry of the productive reaction intermediate for the acetylation of



2-phenylcyclohexanamines catalyzed by CALB [11f].



14.5

Lipase - Catalyzed Polyester Formation



The catalytic reaction of lipases follow the so called ping-pong bi-bi mechanism,

a double displacement mechanism. This is a special multisubstrate reaction in

which, for a two -substrate, two -product (i.e., bi-bi) system, an enzyme reacts with

one substrate to form a product and a modified enzyme, the latter then reacting

with a second substrate to form a second, final product, and regenerating the

original enzyme (ping-pong).

In an attempt to apply this mechanism to a polyester formation and to investigate the single steps involved on a molecular basis one can for instance start with

the CALB –phosphonate complex 1LBS [6] as the initial structure, as it is structurally close to the intermediate structure of an acylated Ser105 that certainly has to

be passed through during an enzymatic polymerization process. In accordance

with the accepted mechanism for serine hydrolases the enzymatic process consists of two mechanistically important steps. These are, in the case of for instance,

a potential enzymatic esterification of adipic acid with 1,6 -hexanediol:







acylation of Ser105 by the diacid (steps a to c in Figure 14.7) which proceeds

via a tetrahedral intermediate structure (Figure 14.7 b)







deacylation of Ser105 by attack of the diol (steps d to f in Figure 14.7) again via

a tetrahedral intermediate structure (Figure 14.7e) regenerating the free Ser105

to be ready for the next elongation step.



It should be noted that the orientation of the molecules in the active site as shown

in Figure 14.7 is not the result of an exact computational simulation but is rather

meant to give an idea of the spatial arrangement of the catalytic triad, the growing

polymer chain and the monomeric building blocks involved. A reliable simulation

of this reaction still remains to be computed.



14.6

CALB - Catalyzed Polymerization of β -Lactam



While there are numerous molecular modeling studies that describe alcoholysis

or aminolysis of acylated serine hydrolases the literature does not yet provide us



357



358



14 Molecular Modeling Approach to Enzymatic Polymerization

(a)



MeO2C

Gln106

N

H

O



Ser105 O

MeO

His224

H

H

COOѲ



N



H



H O

N



Thr40



N



Asp187



(b)



MeO2C

Gln106

N

H



Ѳ

H O

O

H

OMe

N



Ser105 O

His224

N



Thr40



N H



COOH

Asp187



(c)



MeO2C

Gln106

N

H



H O

O H

N



Ser105 O

His224

H

COOѲ



N



Thr40



N

Me OH



Asp187



(d)



MeO2C

Gln106

N

H



Ser105 O

His224

H

COOѲ



N



O

H



N



H O

O H

N



Thr40

OH



Asp187



(e)

MeO2C

Gln106



O



Thr40



N H



N

Asp187



H O

O H

N



Ѳ



Ser105 O

His224



N

H



OH



COOH



(f)

MeO2C

Gln106

N

H



Ser105 O

H



His224

H

COOѲ



N



O



H O

O H

N



Thr40



N

OH



Asp187



Screenshots of a hypothetical CALB - catalyzed esterification with acylation of

serine by adipic acid (steps a to c) and deacylation by 1,6 -hexanediol (steps d to f). Yellow

lines depict hydrogen bondings.



Figure 14.7



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