Chemistry Reference
In-Depth Information
In analogy to the above experiments with pyridoxamine complexes, the 1,10-phenan-
throline ligand was introduced at several alternative sites (positions 60, 72 and 104, see
Figure 5.8) using the protein scaffold IFABP [59]. Using alanine isopropyl ester as a sub-
strate, IFABP-Phen60 catalyzed ester hydrolysis with less selectivity than ALBP-Phen,
while Phen72 promoted the same reaction with higher selectivity. In contrast, hydrolysis
of tyrosine methyl ester was catalyzed with higher selectivity by Phen60 and more rapidly
by Phen104. These results indicated that the rate enhancement and substrate selectivity
of hydrolysis reactions catalyzed by phenanthroline conjugates depended largely on the
orientation and environment of the metal ligand within the protein cavity.
5.3.10
A Flavin-containing Conjugate
Reactions catalyzed by a flavin analog incorporated into IFABP have also been studied
by alkylating a Cys residue within the cavity of the helixless variant (hsIFABP) [60]. The
conjugate hsIFABP-FL catalyzed the oxidation of several dihydronicotinamides. These
experiments were performed mainly to compare results obtained with flavin-IFABP
conjugates with similar results acquired with flavopapains; these latter proteins were
among the first semisynthetic enzymes produced. Interestingly, while hsIFABP-FL
and flavopapain gave comparable rate accelerations (k
cat
/K
M
) for dihydronicotinamide
oxidation, hsIFABP-FL manifested much higher K
M
and k
cat
than did flavopapain. The
low K
M
observed with flavopapain indicates that it primarily accelerates the reaction
rate by enhancing substrate binding, whereas the higher k
cat
obtained with hsIFABP-
FL suggests that its major mode of rate acceleration involves enhancing flavin reac-
tivity. Molecular modeling of hsIFABP-FL indicates that Gln
27
is close to N(3)H and
O(4) of the flavin (Figure 5.15). Interaction between the carboxamide of Gln
27
and the
flavin is likely to alter the redox potential of the isoalloxazine and hence augment its
reactivity. Taken together, these results with flavopapain and hsIFABP-FL highlight
the diverse ways in which protein scaffolds can be used to modulate catalyst activity.
5.3.11
Some Limitations
While the examples described above highlight the impressive results that can be
achieved with protein as scaffolds for catalyst design, this approach is not without pro-
blems. Firstly, there is the issue of cavity size. It would be useful to incorporate large
metal-ligand systems into this protein system to generate catalysts for a plethora of
Figure 5.15 Hydrogen bonding to the flavin in hsIFABP-FL.
