Chemistry Reference
In-Depth Information
Figure 5.9 Multiple turnover reactions that produce chiral amino
acids from achiral keto acids.
turnover conditions suggested that this construct might be capable of catalytic trans-
amination. Using tyrosine or phenylalanine (
5.9-2
) as the amine source to recycle the
cofactor from the pyridoxal form (
5.7-3a
) back to the pyridoxamine form (
5.7-1
), L-glu-
tamate (
5.9-3
) was formed with an enantiomeric purity of 93% ee. As many as 50
turnovers were observed with long reaction times (14 days). Both k
cat
and K
M
for
the reaction were determined by kinetic analysis (Table 5.1).
Comparison with similar parameters obtained from reactions with free pyridoxa-
mine indicated that IFABP-PX60 catalyzed transamination some 200 times more effi-
ciently. Analysis of the specific kinetic constants k
cat
and K
M
indicated that the observed
rate acceleration was due mostly to an increase in substrate binding (50-fold), with a
smaller effect on the maximal rate (4-fold). While this is an impressive result, the ab-
solute magnitude of k
cat
/K
M
(0.02 s
-1
M
-1
) makes it clear that this catalyst is still quite
primitive compared to natural enzyme systems that occasionally operate with catalytic
efficiencies near the diffusion limit.
Table 5.1 Kinetic constants and catalytic efficiencies for semi-synthetic transaminases based on fatty acid
binding proteins.
k
cat
(h
-1
)
k
cat
/K
M
(h
-1
m
M
-1
)
K
M
(m
M
)
10
-4
PX
73
0.032
4.4
10
-4
MPX
38.7
0.031
8.0
IFABP-PX60
1.8
0.29
0.16
IFABP-MPX60
6.8
0.23
0.034
hsIFABP-PX60
10.2
0.22
0.022
IFABP-PxK38
0.81
0.44
0.54
IFABP-MPxK38
13.7
1.12
0.08
IFABP-PxK51
0.24
0.44
1.83
IFABP-MPxK51
8.9
0.52
0.06
IFABP-Px126
5.5
0.18
0.03
IFABP-MPx126
40
1.10
0.03
IFABP-Px126/14
6.0
0.11
0.02
IFABP-MPx126/14
48
0.78
0.02
