Chemistry Reference
In-Depth Information
hsIFABP-PX60 (0.22 h
-1
), while the former exhibited a 4-fold increase in K
M
[56]. After
introducing the MPX cofactor into the cavity of helixless protein, 4.9 turnovers in 24 h
with 35% ee was observed for the production of Glu under the same conditions. Taken
together, these results suggested that removal of the
-helical lid did not affect the
maximal rate and enantioselectivity of the respective constructs. However, deletion
of this structural element did decrease the substrate binding affinity, as evidenced
by an increase in K
M
. Thus, it appears that while the hsIFABP scaffold was not useful
in increasing the efficiency of PX-promoted transamination, these experiments did
show that stable constructs could be prepared based on the helixless scaffold. This
may have greater utility in reactions with larger cofactor catalysts or substrates.
a
5.3.9
Hydrolytic Reactions
Fatty acid binding proteins have also been used as scaffolds for constructing hydrolytic
catalysts. Phenanthroline was attached to Cys
117
of ALBP using iodoacetamido 1,10-
phenanthroline and metallated to produce a coordinated Cu(
II
) ion encapsulated with-
in a chiral protein cavity (Figure 5.14). The resulting conjugate, ALBP-Phen-Cu(
II
) was
able to promote the hydrolysis of several unactivated amino acid esters under mild
conditions (pH 6.1, 25
8
C) at rates 32- to 280-fold above the background rate in buffered
aqueous solution; these reactions also showed modest stereoselectivity [57]. In 24 h
incubations, 0.70-7.6 turnovers were obtained with enantiomeric excesses ranging
from 31 to 86% ee. ALBP-Phen-Cu(
II
) could also catalyze the hydrolysis of an activated
amide (picolinic acid methyl nitroanilide, PMNA), under slightly more vigorous con-
ditions (37
8
C) and after longer incubation times. The rate of amide hydrolysis was 1.6
10
4
-fold higher than the background rate [57]. However, k
cat
obtained with ALBP-
Phen-Cu(
II
) was still significantly lower than that obtained with a related Cu(
II
) bipyr-
idine complex [58]. This rate decrease may reflect a non-optimal, perhaps nonplanar,
conformation for PMNA binding within the ALBP cavity. Of related interest, an X-ray
crystal structure of ALBP-Phen was obtained. Inspection of this structure showed that
the protein could not accommodate the phenanthroline and PMNA within a planar
conformation without significant distortion of the protein backbone [45]. This may
account for the lower than expected rate of PMNA hydrolysis promoted by ALBP-
Phen-Cu(
II
) compared with non-proteinaceous model complexes.
Figure 5.14 Reaction used to prepare ALBP-Phen.
