C O M M U N I C A T I O N S
Table 2. Kinetic Constants for the Michael Addition of
Acetylacetone to Methyl Vinyl Ketone (1 + 4) Catalyzed by C.
antarctica Lipase B Ser105Ala in Cyclohexane at Various
Acetylacetone Concentrations
Table 1. Specific Rates (v) for the Solvent-Free Michael Addition
of the Substrates Shown in Scheme 1a
1
1
1
1
exp.
v
mt (s-
)
v
wt (s-
)
k
non (s-1 M-
)
v )
mt/knon (M-
1 + 3
1 + 4
1 + 5
2 + 3
2 + 4
2 + 5
4.0 × 103
1.2 × 100
8.1 × 10-2
<10-7
1.1 × 102
9.0 × 10-1
2.4 × 10-3
<10-7
2.6 × 10-5
4.8 × 10-9
2.6 × 10-8
<10-10
1.5 × 108
2.4 × 108
3.1 × 106
concentration (1)
app
0.10 M
0.50 M
1.0 M
k
k
/KaMpp (s-1 M-1
)
1.2
1.0
0.66
cat
non (s-1 M-1
)
4.8 × 10-9
4.8 × 10-9
4.8 × 10-9
(kapp/KaMpp)/knon
2.4 × 108
2.2 × 108
1.4 × 108
3.2 × 10-1
5.8 × 10-3
4.4 × 10-7
7.2 × 105
cat
9.0 × 10-3
1.1 × 10-5
1.5 × 10-11
5.9 × 108
a Specific rates (V) were calculated from initial rates as mole product
per mole enzyme and second. The knon values were calculated from the
reaction rates, V ) knon [S]1[S]2, where [S]1 and [S]2 are the concentrations
of the two substrates.
Kinetic constants for the Michael addition catalyzed by the
Ser105Ala mutant of C. antarctica lipase B were determined for
methyl vinyl ketone 4 under pseudo-one substrate conditions,
keeping the concentration of acetylacetone constant (Table 2). The
values of kcaaptp/KMapp (∼1 s-1 M-1) were calculated from initial rate
determinations at a low concentration of methyl vinyl ketone 4 (0.04
M). The experiment was repeated at three concentrations of
acetylacetone 1 (0.1, 0.5, and 1.0 M). The decrease in kacaptp/KMapp
with increased concentration of acetylacetone is probably caused
by binding of that substrate to the oxyanion hole, competing with
methyl vinyl ketone. The catalytic proficiency ((kacaptp/KMapp)/knon
)
was over 108, which is in the same order as the value observed for
enzymes with native substrates.12
In conclusion, we have increased the reaction specificity of C.
antarctica lipase B Ser105Ala toward carbon-carbon bond forming
Michael additions. The specific rates for the tested substrates were
high and even extremely high for the Michael addition of acetyl-
acetone to acrolein. Data from Michael addition reactions catalyzed
by C. antarctica lipase B Ser105Ala showed saturation kinetics
with substrate inhibition of both substrates. The rate enhancement
(105-108) and the catalytic proficiency (>108) were in the same
range as the values observed for enzymes with native substrates.
Figure 2. The progress curve of the solvent-free Michael addition of
acetylacetone to acrolein 1 + 3 in Table 1. CALB ) C. antarctica lipase
B.
The ability of wild-type and Ser105Ala mutant of C. antarctica
lipase B to catalyze the Michael addition of 1,3-dicarbonyl
compounds to R,â-unsaturated carbonyl compounds was tested
under solvent-free conditions (Table 1). All experiments proceeded
faster with the mutant than with the wild-type enzyme (1.3-830
times). The reaction of acetylacetone with acrolein 1 + 3 (Table
1) proceeded extremely fast with the mutant reaching 100%
conversion in less than 10 min (Figure 2). The specific rate (4000
s-1) was found to be 36 times higher than the specific rate for the
wild-type lipase. This reaction has previously been catalyzed by
CeCl3‚7H2O/NaI without solvent, which resulted in 86% yield after
8 h.11 On the basis of mole catalyst, the enzymatic process is 107
more efficient. Methyl acrylate 5 showed the lowest specific rates
of the three R,â-unsaturated carbonyl compounds tested. Dimethyl
malonate 2 reacted slower than acetylacetone 1 in all reactions
(Table 1). This can be due to the more acidic R-protons of
Acknowledgment. This work was supported by The Swedish
Research Council (VR).
References
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is increased, the reaction will start to boil. The progress curve of
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2. This curve shows the progression of the reaction catalyzed by
the mutant enzyme, wild-type enzyme, and two uncatalyzed
reactions. The two uncatalyzed reactions (one with carrier without
enzyme and one with substrates only) showed low reaction rates.
This indicates that the Michael addition is catalyzed by the enzyme.
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