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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA072444-04
Application #
6172924
Study Section
Biochemistry Study Section (BIO)
Program Officer
Lees, Robert G
Project Start
1997-09-15
Project End
2002-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
4
Fiscal Year
2000
Total Cost
$352,909
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
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Ho, Meng-Chiao; Sturm, Matthew B; Almo, Steven C et al. (2009) Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins. Proc Natl Acad Sci U S A 106:20276-81
Luo, Minkui; Schramm, Vern L (2008) Transition state structure of E. coli tRNA-specific adenosine deaminase. J Am Chem Soc 130:2649-55

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