Chemistry Reference
In-Depth Information
Figure 5.12 Putative binding site for the side-chain carboxylate of the
substrate
-keto glutarate in IFABP-PX60. Present are Y
14
(upper left), R
126
(upper right) and the Schiff base complex between
a
-keto glutarate and
the pyridoxamine catalyst. (lower center). Dashed lines indicate putative
hydrogen bonds between the
a
-keto glutarate and the
side-chain phenol of Y
14
and the guanidinium group from R
126
. Color
scheme: Carbon (white), oxygen (red), nitrogen (blue), sulfur (orange).
c
-carboxylate of
a
Computer modeling together with mutagenesis was also used to identify existing
residues in the protein cavity that contributed to the substrate specificity of the IFABP-
PX60 catalyst. R
126
and Y
14
were identified as two important residues that interacted
with the
-keto glutarate when bound to the enzyme via a
Schiff base with the PX moiety (Figure 5.12). Mutants IFABP-V
60
C,R
126
M and
-V
60
C,R
126
M,Y
14
F were prepared and pyridoxamine was attached to each mutant
[53]. Of particular note, IFABP-PxM126 had a K
M
three-fold higher than IFABP-
PX, while the second mutation at position 14 had no significant effect. The k
cat
s
for both conjugates were 2-fold lower than for the original IFABP-PX60. N-methylated
pyridoxamine conjugates were also introduced into these two mutants. Compared to
the original IFABP-MPX60 conjugate described above, the mutations in positions 126
and 14 increased K
M
as much as seven-fold. IFABP-MPxM126 gave a particularly
significant result, manifesting a 5-fold higher k
cat
. While neither the experiments
with the lysine mutants nor those with the carboxylate binding site mutants produced
catalysts that rival the efficiency of natural enzymes, incremental improvements were
observed with many of the conjugates. Moreover, these advances came from con-
structs that combined mutations in the protein scaffold with synthetic alterations
in cofactor structure, thus highlighting the power of the chemogenetic approach.
c
-carboxylate group of
a
