A Procedure for the Joint Evaluation of Substrate
Partitioning and Kinetic Parameters for Reactions
Catalyzed by Enzymes in Reverse Micellar Solutions
I. Hydrolysis of 2-Naphthyl Acetate Catalyzed by Lipase in Sodium
1,4-Bis(2-ethylhexyl) Sulfosuccinate (AOT)/Buffer/Heptane
Luis Felipe Aguilar,
1
Elsa Abuin, and Eduardo Lissi
Facultad de Quı ´mica y Biologı´a, Universidad de Santiago de Chile, Casilla 40, Correo 33, Santiago, Chile
Received September 18, 2000, and in revised form January 12, 2001; published online March 14, 2001
A simple method useful for the joint evaluation of
substrate partitioning and kinetic parameters for re-
actions catalyzed by enzymes entrapped in reverse
micelles is proposed. The method is applied to the
hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by
lipase in sodium 1,4-bis(2-ethylhexyl) sulfosuccinate
(AOT)/buffer/heptane reverse micellar solutions. In
the presence of micelles, the relationship between the
initial reaction rate and the analytical concentration
of 2-NA was dependent on AOT concentration at a
constant W ([water]/[AOT]) value. The dependence of
the initial reaction rate profiles with [AOT] was ana-
lyzed according with the method proposed to obtain
the partition constant of 2-NA between the micelles
and the external solvent, K
p
. A value of K
p
2.7 L mol
1
was obtained irrespective of the water content of the
micelles (W from 5 to 20). The catalytic rate constant
k
cat
in the micellar solutions was independent of [AOT]
but slightly decreased with an increase in W from 2
10
6
mol g
1
s
1
at W 5 to 1.2 10
6
mol g
1
s
1
at W
20. The apparent Michaelis constant determined in
terms of the analytical concentration of 2-NA in-
creased with [AOT] at a given W and moderately de-
creased with W at a fixed [AOT]. The increase with
[AOT] is accounted for by considering the partitioning
of the substrate. After correction for the partitioning
of 2-NA values of (K
m
)
corr
were obtained as 3.9 10
3
mol L
1
(W 5), 4.6 10
3
mol L
1
(W 10), 2.3 10
3
mol L
1
(W 15), and 1.7 10
3
mol L
1
(W 20). The
rate parameters in the aqueous phase in the absence
of micelles, were obtained as (k
cat
)
aq
7.9 10
6
mol g
1
s
1
and (K
m
)
aq
2.5 10
3
mol L
1
. In order to compare
the efficiency of the enzyme in the micellar solution
with that in aqueous phase, the values of (K
m
)
corr
were
in turn corrected to take into account differences in
the substrate activity, obtaining so a set of (K
m
)
*
corr
values. The efficiency of the enzyme in the micellar
solution, defined as the ratio, k
cat
/(K
m
)
*
corr
, was found to
be higher than in the aqueous phase, even at high water
contents (W 20). This higher efficiency is due to a
significant decrease in (K
m
)
*
corr
values. © 2001 Academic Press
Key Words: enzyme kinetics; lipase; reverse micelles.
Since 1977, after the pioneer work of Martinek et al.
(1), micellar enzymology has arisen as a new physico-
chemical line of research to approach problems in mo-
lecular biology. Micellar enzymology is mainly devoted
to the study of reactions catalyzed by enzymes in re-
verse micellar solutions. The field has attracted the
interest of many researchers, but in spite of more than
two decades of intensive work, several key problems
remain unresolved, particularly in the area of enzyme
kinetics.
Many studies of enzyme kinetics in reverse micellar
solutions (RMS)
2
have been reported (2–26). In most of
them, the enzyme is totally associated to the micelles
while the substrate is partitioned between the micelles
and the external solvent. Concerning the kinetic be-
havior, a relevant question is whether the efficiency of
the enzymes in the RMS is different from that in bulk
aqueous solution. To answer this question requires a
1
To whom correspondence should be addressed. Fax: 56-2-
6812108. E-mail: luis.aguilar.c@mail.ucv.cl.
2
Abbreviations used: RMS, reverse micellar solution; 2-NA,
2-naphthyl acetate; AOT, sodium 1,4-bis(2-ethylhexyl) sulfosucci-
nate; Lip, lipase; 2-N, 2 naphthol.
0003-9861/01 $35.00 231
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
Archives of Biochemistry and Biophysics
Vol. 388, No. 2, April 15, pp. 231–236, 2001
doi:10.1006/abbi.2001.2289, available online at http://www.idealibrary.com on