Chemistry Reference
In-Depth Information
Figure 5.8 Stereo view showing positions 60, 72, 104 and 117 in
IFABP that have been used for the attachment of pyridoxamine and
other catalytic groups. From top to bottom: A104, Y117, L72 and V60.
Color scheme: Protein secondary structure (green), carbon (white),
oxygen (red), nitrogen (blue).
The three different mutant proteins described above were purified and the corre-
sponding conjugates were prepared using the TP-PX reagent (
5.6-1
). The conjugates
were then evaluated in single turnover conditions (Figure 5.7) with several
-keto acids
to produce amino acids in enantiomerically enriched form. This collection of catalysts
exhibited various differences in reactivity and selectivity. Compared to ALBP-PX,
IFABP-PX60 reacted at least 9.4-fold more rapidly, while IFABP-PX72 displayed op-
posite enantioselectivity, and IFABP-PX104 showed a clear selectivity preference for
unbranched substrates [47]. From these experiments, the position of cofactor attach-
ment is clearly an important parameter in modulating the reactivity and specificity of
these biocatalysts. Moreover, these results underscore the utility of site-directed mu-
tagenesis and the power of using a protein-based scaffold for catalyst development.
a
5.3.4
Catalytic Turnover with Rate Acceleration
Due to its intriguingly more rapid reaction rate than ALBP-PX and free pyridoxamine,
IFABP-PX60 was subsequently studied further [48]. Under single turnover conditions,
it converted
-keto glutarate (Figure 5.9,
5.9-1
) into glutamic acid (
5.7-4
) 62-fold faster
than free pyridoxamine (
5.7-1b
); this was determined by evaluating the extent of con-
version at much shorter reaction times. This more rapid reaction rate under single
a
