RICIN--MECHANISM, TRANSITION STATE AND INHIBITOR DESIGN

Project: Research project

Project Details

Description

Ricin is an RNA-depurinating toxin found in the castor bean. A single
adenine is hydrolyzed from 28S rRNA, rendering the ribosome
inactive. One molecule is lethal for mammalian cells, making it one of
the most toxic biological molecules. The cytotoxicity of ricin is now
being exploited in clinical trials to destroy unwanted cells. Despite the
novel catalytic properties and potential clinical uses of ricin, little is
known of it's substrate specificity, catalytic mechanism or transition
state structure. The only inhibitors with Km/Ki greater than 1 have
been prepared by this laboratory.

Recent advances in the application of kinetic isotope effects to
enzymatic reactions has permitted the characterization of the major
features of several N-ribohydrolase enzymatic transition states. Ricin
A-chain catalyzes a reaction chemically similar to nucleoside and
nucleotide N-ribohydrolases and is a candidate for similar analysis.
Availability of transition state information proves fundamental
information of the catalytic mechanism and has assisted in chemical
mechanisms and transition state structure for the catalytic subunit of
ricin, ricin A chain. This information will be applied to the design of
molecules which are inhibitors of the enzyme. The proposed inhibitors
will be synthesized and characterized by kinetic and binding
experiments. Stem-loop RNA and hybrid inhibitor molecules will be
synthesized to define substrate and inhibitor specificity. Selected stem-
loop RNA structures will be solved by NMR. Incorporation of a spin-
label at the depurination site of stem-loop RNA analogues will be used
to provide a binding probe and to permit mapping of the protein-RNA
geometry in the catalytic site cavity. The transition state structure will
be investigated by kinetic isotope effects. Substrate RNA analogues
will be synthesized to test if solvolysis of the N-ribosidic bond depends
on leaving group activation (acid-catalyzed solvolysis), ribosyl activation
(ribooxocarbenium ion stabilization) or ribosyl hydroxyl ionization
(base-catalyzed solvolysis) or a combination of more than one of these
mechanisms. Inhibitors which bind tightly will be characterized by
cocrystalization with ricin A-chain for x-ray crystal studies.
StatusFinished
Effective start/end date9/15/976/30/98

Funding

  • National Cancer Institute

ASJC

  • Catalysis

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