Chemistry Reference
In-Depth Information
tions (see below for further discussion of those proteins). In those studies, efforts were
made to increase their catalytic efficiency by employing a genetic method to enforce
N-protonation. Taking a cue from the crystal structure of AATase (aspartate amino
transferase), an enzyme that catalyzes transamination using a pyridoxamine cofactor,
carboxylate-containing amino acid residues were introduced close to the pyridine
nitrogen locus in two IFABP mutant proteins. It was thought, based on functional
studies of AATase, that the positioning of anionic side chains near the pyridine nitro-
gen center would enforce protonation via an ion pairing mechanism; the presence of
the proximal negative charge would favor the protonated pyridine. Several carboxylate-
containing IFABP mutants were prepared for this purpose. Unfortunately, none of
these mutants were sufficiently stable for conjugate preparation - they precipitated
during purification, and efforts to refold the denatured protein were unsuccessful.
However, in separate experiments, the TP-MPX reagent (
5.6-3
) was used to prepare
MPX-containing constructs instead (see
5.6-4
for a generic structure). Interestingly,
these conjugates showed enhanced catalytic activity compared to their PX progenitors
[51]. Kinetic studies indicated that the k
cat
(1.12 h
-1
for IFABP-MPxK38 and 0.52 h
-1
for
IFABP-MPxK51) and turnover numbers (12.2 turnovers by IFABP-MPxK38 and 5.7 by
IFABP-MPx51 in 24 h) observed with these constructs under standard conditions are
the highest achieved in this system (see Table 5.1 for a summary of kinetic para-
meters). The success of these constructs, prepared using a combination of chemical
modification of the catalyst structure and genetic manipulation of the protein scaffold,
highlights the enormous power and flexibility of this chemogenetic approach for cat-
alyst development.
5.3.7
Adding Functional Groups within the Cavity
A major goal of research involving these protein scaffolds was to determine whether
the flexibility of using a protein scaffold could be fully capitalized upon by using ra-
tional design in concert with knowledge of chemical mechanism to improve catalytic
efficiency. Taking a cue fromNature, based on biochemical experiments with AATase,
lysine residues were introduced into the protein cavity to enable Schiff base formation
and to serve as general acids and bases in the reaction cycle. Figure 5.10 shows these
functions in an abbreviated transamination mechanism.
Based on the crystal structure of IFABP, molecular modeling was employed to iden-
tify possible positions where lysine residues could be introduced to perform the func-
tions noted above. Mutants L
38
K,V
60
C and E
51
K,V
60
C were prepared and used to create
pyridoxamine conjugates [52]. Figure 5.11 gives a model of IFABP-PXK51. The result-
ing assemblies IFABP-PxK38 and IFABP-PxK51 showed improved k
cat
and K
M
. The
overall catalytic efficiency (k
cat
/K
M
) of IFABP-PxK51 increased 4200-fold compared
to unliganded pyridoxamine phosphate and was 12-fold greater than for IFABP-
PX60 while maintaining comparable enantioselectivity (83-94% ee). The principal
effect on the kinetic constants for the reactions catalyzed by these mutants was on
K
M
. Each conjugate showed a significant decrease in K
M
(2.2- and 7.5-fold) and a small
increase in k
cat
(1.5-fold). UV/Vis spectroscopy, fluorescence and electron-spray mass
