Chemistry Reference
In-Depth Information
interesting reactions. Unfortunately, the enclosed cavity of fatty acid binding proteins
(and even the somewhat open cavity of hsIFABP) limits the size of what can be in-
troduced into the protein interior. Efforts to incorporate a heme group or a Mn(
III
)-
salen complex into hsIFABP were not successful. While it was possible to attach these
moieties to the protein scaffold under denaturing conditions and refold them, the re-
sulting conjugates were unstable and underwent aggregation and precipitation upon
storage. In future work, it will be necessary to identify other protein structures that can
serve as effective scaffolds but that are not subject to these size limitations. Secondly,
work with fatty acid binding proteins is limited to aqueous systems. This creates two
types of problems. The first is that catalytic systems that are sensitive to water cannot
be employed. The Mn(
III
)-salen complex described above underwent Schiff base hy-
drolysis and subsequent ligand decomposition upon prolonged storage in aqueous
buffer. While the rate of this hydrolytic degradation can be decreased by employing
mixtures of water and organic solvents, fatty acid binding proteins have limited solu-
bility in such solvents. In addition, the inability to work in such solvents also limits the
substrate concentrations that can be used and also precludes the use of these catalysts
with many types of hydrophobic substrates. However, given the extensive interest and
the large body of literature concerning the use of enzymes in organic media, it should
be possible to identify other types of proteins that can serve as scaffolds for catalyst
design in such solvent systems.
5.4
Myoglobin as a Starting Point for Oxidase Design
5.4.1
Artificial Metalloproteins and Myoglobin
Metalloproteins represent a major faction of scaffold proteins in artificial enzyme de-
sign. The introduction of a wide range of metals and redox cofactors into protein scaf-
folds can greatly increase both the diversity of enzyme catalysis and the application of
these artificial metalloproteins. To sequester artificial redox cofactors into protein scaf-
folds, which would otherwise not bind these chemical catalysts, one may either use
non-covalent or covalent attachment [61]. Myoglobin is a well-studied protein that con-
tains a non-covalently bound heme for oxygen transport. The large heme-binding site
provides substantial room to accommodate other ligand systems, and for these reasons
serves as a useful starting point for catalyst design.
5.4.2
Non-covalent Attachment of a Redox Center
Watanabe and co-workers pursued an approach involving the non-covalent placement
of Mn (III) and Cr (III) salophen complexes into apo-myoglobin [61]. In this artificial
metalloprotein, two residues required mutation to improve the binding affinity of the
