Biology Reference
In-Depth Information
Computational Redesign of Enzyme Reactivity
The redesign of enzyme reactivity, while still considered redesign, represents a more drastic
intrusion into the redesigned enzyme than mere specificity redesign. In this approach, the
active site of an existing enzyme is redesigned, or
to catalyze a different type of
reaction. Arguably, the more similar the two reaction types are the easier the task becomes.
A class of enzymes inherently suited for this approach is metalloenzymes. In many
metalloenzymes, the metal carries out the essential chemical step, while the surrounding
active-site amino acids serve to bind and orient the metal and the other (usually organic)
substrate(s) in the proper orientation to each other, while also activating relevant functional
groups of the organic substrate. Depending on what type of organic functional group is
bound proximal to the metal, the same metal center can catalyze different chemistries. For
example, metals such as zinc have an inherent affinity for water molecules. An H
2
O
molecule has reduced pKa when bound to zinc, and the resulting ZnOH species is a more
potent nucleophile than unactivated H
2
O. Depending on the electrophile present, the zinc
bound OH
2
could then either undergo a hydrolytic or a nucleophilic addition reaction.
'
repurposed,
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In the inaugural example of this approach, Khare et al. redesigned an adenosine deaminase
into an organophosphate hydrolase.
4
The wild-type enzyme, which was part of a set of
enzymes featuring mononuclear Zn-sites with at least one of the Zn-coordination sites not
occupied by a side-chain, contains a Zn-binding site that activates a water molecule for
nucleophilic attack onto the amino group of adenosine. In their work, the authors first
placed the design substrate, a model organophosphate featuring an activated leaving group,
in the active site such that the Zn was coordinating to the phosphate
s keto-oxygen, thus
rendering the phosphorus atom more susceptible to nucleophilic attack by a water
molecule. Next, the side-chain placement algorithm was used to redesign the surrounding
active site residues to accommodate the new substrate. During this step, the Zn-coordinating
residues were held constant. The resulting design featured eight mutations and hydrolyzed
the model substrate with a k
cat
/K
M
of 4 M
2
1
s
2
1
, and after three rounds of directed
evolution, a variant with a total of 13 mutations and a k
cat
/K
M
of
'
113
10
4
M
2
1
s
2
1
for the
designed substrate was obtained. Further analysis of the importance of each of the
mutations indicated that a minimal set of four mutations was absolutely required to confer
hydrolytic activity for the target substrate. This suggests that obtaining a comparable result
through directed evolution alone would necessitate screening of an enormous library
containing all possible quadruple mutations of the protein
B
s active site.
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Computational De Novo Design of Enzyme Activity
The third and most difficult type of problem in computational enzyme design is the design
of catalytic activity from scratch. In this case, both a catalytic mechanism as well as a protein
site to carry it out need to be devised. The currently most viable approach, depicted in
Figure 6.2
, consists of developing a so-called theozyme for the reaction of interest, and then
trying to graft this theozyme into a scaffold protein. A theozyme, or
theoretical enzyme,
is
'
'
a three-dimensional model of a minimal active site necessary to catalyze the desired
reaction.
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Usually it consists of a model of the energetically highest transition state on the
reaction pathway, together with a set of disembodied amino acids placed around it that are
meant to stabilize this state or perform chemical transformations on it. For example, if
negative charge develops on a certain substrate atom over the course of the reaction, a
strategy to stabilize this build-up and thus accelerate the reaction would be to place a
positively charged side-chain, such as an arginine or lysine, next to this substrate atom. If
the substrate gets deprotonated, a protic amino acid, such as a glutamate, aspartate, or
histidine could be placed next to the mobile proton in the theozyme. Several strategies to
develop a theozyme for a given reaction of interest have been devised. The most
comprehensive and most difficult one represents an approach using quantum-mechanical
