AREAS RELATED TO ENZYME CATALYSIS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 169

5.4. Enzymes design and redesign

Recent advantages in enzyme catalysis, protein chemistry and sequences, and the
determination of three-dimensional structures and genetic engineering have laid a basis
for the development of methods for enzyme design and redesign (Ferst, 1999; Clelend
and Craik, 1996; Altamarino et al., 2000; Benson et al., 2000; Babitt, 2000; Ness et al.,
2000; Ostermeier aand Bencovic, 2000; Fersht and Alamarino, 2001; DeGrado, 2001;
Penning and Jetz, 2001; Lu et al., 2001; Oi et al., 2001; Tann and Oi, 2001; Tann et al.,
2001; Saven, 2001; Arnold, 2000, 2001; Arnold and Volkov, 1999). The following
directions in this area have sparked interest: 1) introducing novel functionality in native
enzymes and protein by modifying their sequence and chemical composition; 2) directed
evolution (mimicking the evolution of analogs in vitro

3) producing semisynthetic
enzymes by attaching new functionality; and 4) the design of protein sequences de novo.
A generation of new enzymes via covalent modification of existing proteins can be
produced using several methods (Oi et al., 2001; an references therein). Chemical
approaches for converting catalytic groups of enzymes have been described . For
instance, the active site serine hydroxyl group of subtilisin was replaced by a thiol and
the active site thiol was changed for a hydroxyl. An alternative approach involves the
replacement of large portions of a protein via proteolysis or chemical cleavage.
Ribonuclease A was cleaved by subtilisin into two fragments, S-peptide and S-protein,
followed by the introduction of a pyridoxal cofactor to S-protein . The modified protein
catalyzes convertion L-alanine to pyruvate. Flavin analogues were incorporated into the
active site groove of papain that was used as the protein scaffold. These new
semisynthetic enzymes catalyze the oxidation of dihydronicotinamides with activity of
about 10% relative activity of the native NADH-specific FMN reductase.
Another protein and enzyme design process bases on the introduction of metal-
binding sites into protein scaffolds (Lu et al., 2001). This approach includes two steps: 1)
the choice of scaffolds such as de novo designed structures; and 2) the design
and engineering of metal-containing active sites. This approach involves the redesign of
existing metal-binding sites to new sites with different functions and the design and
engineering of new metal-binding sites. In the frame of the first direction, experiments
on the variation of proxymal and distal ligands and types of cofactors of heme proteins
were performed. The most interesting results of these experiments were the successful
transformation of heme-histidine proteins to heme-cysteine enzymes analogues, such as
cytochrome P450 and chloroperoxidase. The human myoglobin with the proximal
cysteine ligand exposes spectral properties typical for active sites of above mentioned
enzymes. In the result of the modification, a 5-fold increase of in P450-like
monooxydegenase activity was observed. Redesign of copper, non-heme iron and other
metal-containing proteins have been also performed (Lu et al., 2001 and references
therein). Design and engineering of new metal-binding sites involves rational design
using the automated computer search algorithm and other empirical and semiempirical
approaches, as well as design by combinatorial /evolution methods (selection of
metalloproteins through phage display, search for metalloantibodies, and directed
evolution of heme enzymes). For instance, the peroxidase activity of horse heart
myoglobin was 25-fold improved using the random mutagenesis technique.

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