Project: Research project

Project Details


Transition state theory for enzymatic reactions indicates that molecules
which resemble the enzyme-bound transition state will capture a fraction
of the transition state binding energy and provide powerful and specific
noncovalent inhibitors. This theory has gained wide acceptance but
applications have been limited by the lack of methods to experimentally
determine reliable transition state structures for enzymatic reactions.
Recent advances in the application of kinetic isotope effects have
permitted unprecedented understanding of the geometry and electronic
nature of enzyme-stabilized transition states.

The inosine-preferring nucleoside hydrolase from the trypanosome Crithidia
fasciculata has provided a test case to establish that transition state
information can be determined with sufficient accuracy to permit the
design of powerful inhibitors. A family of inhibitors is now available to
establish the interactions between the enzyme and the transition state.
The inhibitors can also be used to explore a surprising diversity of
transition state structures which exist in the nucleoside hydrolases.

Nucleoside hydrolases are found in protozoa including Trypanosoma (Chagas'
disease and sleeping sickness), Leishmania (leishmaniasis), Entamoeba
(amebic dysentery), Plasmodium (malaria), Giardia (giardiasis) and
Trichomonas (vaginitis). Protozoan parasites are incapable of de novo
purine synthesis and require purine salvage for DNA and RNA synthesis.
Mammalian tissues lack nucleoside hydrolases. Transition state inhibitors
for inosine-preferring nucleoside hydrolase are highly specific and do not
inhibit mammalian purine nucleoside phosphorylase or guanosine-preferring
nucleoside hydrolase from C. fasciculata.

The hypothesis for this proposal is that transition state inhibitors for
nucleoside hydrolases are isozyme- and Protozoan-specific. The basis of
this specificity will be established by transition state analysis of the
guanosine-preferring nucleoside hydrolase from C. fasciculata. At least
one additional nucleoside hydrolase will be obtained from cDNA and genomic
libraries of Trypanosoma or Plasmodium. Inhibitors will be designed based
on the geometric and electrostatic potential surface of the transition
state. The genes for nucleoside hydrolases will be isolated from cDNA and
genomic libraries and overexpressed in E. coli. The basis for specificity
will be established by X-ray crystallography, pH profiles and transition
state specificity studies. The results will provide novel information on
transition state structure, catalysis, and transition state inhibitors
which may be antimetabolites for protozoan parasites.
Effective start/end date8/1/947/31/95


  • National Institute of General Medical Sciences


  • Infectious Diseases

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