Chemistry Reference
In-Depth Information
5.3.5
Modulation of Cofactor Reactivity with Metal Ions
Model studies with pyridoxamine and related analogs have shown that transamination
reactions promoted by these catalysts can be accelerated by the addition of metal ions,
including Zn(
II
), Cu(
II
), Ni(
II
) and Al(
III
). Hypothetically, these cations accomplish this
by stabilizing the formation of Schiff base intermediates and by increasing the acidity
of the protons that must be removed in the reaction. Interestingly, the addition of
metal ions to reactions catalyzed by ALBP- and IFABP-pyridoxamine conjugates re-
sulted in both positive and negative reaction rate perturbations [49]. IFABP-PX104
reacted 4.7-fold faster in the presence of Cu(
II
) whereas IFABP-PX60 reacted
4.4-fold slower - both rate effects were accompanied by a decrease in reaction enan-
tioselectivity. Little change was observed for the reaction catalyzed by IFABP-PX72.
UV/Vis spectroscopy experiments that monitored the formation of metal-aldimine in-
termediates suggested that IFABP-PX60 and IFABP-PX104 but not IFABP-PX72
formed a complex with Cu(
II
). Thus, metal ions may be used to increase semisynthetic
enzyme efficiency, although this did not occur in all cases. These studies also noted
that the reactions rates were sensitive to changes in buffer; the use of imidazole
resulted in a lower rate than with similar reactions performed in HEPES buffer. These
observations raise the possibility that buffer molecules may actually enter the protein
cavity and directly participate in the reaction. This makes sense because several proton
transfers must occur in a complete transamination reaction and yet there are no func-
tional groups within the cavity that could do this.
5.3.6
Chemogenetic Approach
In the catalytic mechanism of pyridoxamine-based reactions, the pyridine nitrogen
atommust be protonated; this protonation gives the pyridine ring a net positive charge
that increases the acidity of the nearby benzylic protons and serves to stabilize a devel-
oping negative charge. One way protonation can be effectively enforced is by introdu-
cing a permanent positive charge via N-methylation. In some model systems, an N-
methylated species accelerates the transamination rate up to 20-fold. In contrast, after
reconstitution with N-methylpyridoxamine, alanine aminotransferase lost its activity
almost completely (
>
99.8%). Therefore, N-quaternization may have both positive and
negative influences on the reaction rate. To allow a similar modification to be made in
these fatty acid binding protein systems, a new reagent containing N-methylpyridox-
amine and an activated disulfide (TP-MPX,
5.6-3
) was prepared. A conjugate, IFABP-
MPX60 (
5.6-4
) was prepared [50] and, under catalytic conditions, significant reaction
(5.2 turnovers, 41% ee) was observed using
-keto glutarate and phenylalanine as sub-
strates. Kinetic analysis of the reaction showed that the MPX-containing conjugate
gave higher K
M
(3.8-fold higher than the earlier PX construct) but similar k
cat
. This
indicated that N-methylation had no positive effects on the reaction catalyzed by con-
jugates based on IFABP-V
60
C. However, the MPX cofactor proved to be considerably
more useful in subsequent experiments with IFABP variants containing lysine muta-
a
