Structural Investigations of Productive Enzyme Complexes

Project: Research project

Project Details


DESCRIPTION (applicant?s abstract): Serine proteases are a class of enzymes
that play critical roles in many human diseases, including cancer, cystic
fibrosis, rheumatoid arthritis, emphysema, thromboembolic disease, hepatitis C,
and cytomegalovirus. The design of highly specific serine protease inhibitors
is a promising therapeutic approach for disease treatment; the potential of
structure-based drug design has been amply demonstrated by the clinical success
of HIV protease inhibitors in combating AIDS. In addition to serving as
pharmaceutical targets, serine proteases also serve as a useful model for
elucidating the strategies that enzymes use for catalysis. Despite all that is
known about serine protease structure and mechanism, no solved structures are
available for these enzymes complexed with their substrates. This missing piece
of information stems from a technical difficulty; crystal structures present a
molecular picture averaged over time, while enzymes complexed with their
targets usually react too quickly to be captured by standard crystallographic
methods. Developments in technology, including powerful synchrotron radiation
sources and cryocrystallographic techniques, now have enabled structure
determination for several other enzymes complexed with their natural
substrates. The first aim of this proposal is to obtain a crystal structure for
a representative serine protease, subtilisin, complexed with a specific
substrate. The second aim is to use the methodology developed for the structure
determination of the first subtilisin-substrate complex to obtain structures
for a series of mutant subtilisin complexes. Structure-reactivity correlations
will be drawn to clarify the importance of substrate orientation in catalysis.
The final aim is to compare in detail the structure of a subtilisin-substrate
complex with that of a subtilisin-protein inhibitor complex, to determine the
structural features that make one complex reactive and the other unreactive.
Because of the conservation of active site geometry and chemical mechanism in
serine proteases, conclusions drawn about the orientation requirements for the
hydrolysis reaction will be generalizable to all serine proteases. Taken
together, the information provided by the successful completion of this
proposal will provide an improved template for the future design of therapeutic
inhibitors of enzymes in the serine protease family.
StatusNot started


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.