Biomedical Engineering Reference
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and lipase from Thermomyces lanuginosum (TLL) (Brzozowski et al ., 2000 ) contain an
active site covered by a peptide loop known as the lid. All of these enzymes require interfa-
cial activation to open the active site (Reis et al ., 2009). Upon formation of a micelle, the
covering lid interacts with lipid-aqueous interface and exposes the active site (Winkler
et al ., 1990; Lowe, 2002). An “open” lipase is additionally stabilized by protein colipase,
which helps to remove the lid from the active site (Thomas et al ., 2005 ). Other lipases, such
as PLRP2 from guinea pig, Candida antarctica lipase B (CALB) (Uppenberg et al ., 1994 )
and lipase A from bacteria Bacillus subtilis (van Pouderoyen et al ., 2001 ), have no lid and
do not require interfacial activation.
All lipases, regardless of their structure and necessity of interfacial activation, follow the
same mechanism of catalysis (Haeffner and Norin, 1999; Reis et al ., 2009 ). The triad of
amino acids, Ser153, Asp177 and His264 (numbering according to hPL, mature protein),
initiate conversion of substrates. The charge-transfer residues Asp and His assist in partial
ionization of Ser by abstracting its proton. The ionized residue E - Ser - O δ − attacks the ester
bond of a substrate, for example triacylglycerol (TAG), and cleaves it into two fragments.
The produced organic alcohol, for example diacylglycerol (DAG), is liberated into the
medium, whereas fatty acid (FA) becomes covalently bound to Ser. At the next stage, the
second substrate R - OH removes fatty acid from the Ser-residue by a substitutive mecha-
nism. In aqueous solution, R - OH corresponds to water which liberates free fatty acid. In
non-aqueous solution, R-OH can be an alcohol, carboxylic acid or acylglycerol. All of them
attack the Ser - FA bond to produce a novel ester.
Reaction kinetics of lipases is relatively complex and generally depends on the reaction
conditions, which can be aqueous, interfacial or non-aqueous. In an aqueous medium, lid-
less enzymes preferentially hydrolyze soluble substrates such as p-nitrophenyl acetate,
triacetin and tributyrin. This type of lipolytic reaction follows the mono-substrate Michaelis-
Menten equation (Reis et al ., 2009). Concentration of the second substrate (water, 55.5 M)
is assumed as a constant and becomes a part of parameters V and K mS .
Lipases with a lid demonstrate a more complex behavior in aqueous medium. They react
preferentially with micellar substrates ( S *). Figure 14.1 shows the mechanism for simulta-
neous hydrolysis of S and S *. The velocity of the reaction is expressed as the sum of
Michaelis and Hill equations where a steep increase of activity is observed after formation
of S * ( s
K * ).
V
V
*
s
v
=
+
(14.9)
K
n
n
S
K
S
*
1
+
1
+
1
+
S
1
+
n
K
s
K
n
S
*
S
V s and V * are the maximal rates of hydrolysis of S and S * ; K s and K * = (K n ·K s * ) 1/ n are the
half-effect constants of the corresponding catalytic reactions ( K n and K s * are the dissociation
K S *
K S
V
V
*
S *
ES *
++
+
ES
SE
PE
E + P
K n
n.S
Figure 14.1 Mechanism for simultaneous hydrolysis of substrate and micellar substrate.
 
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