Recent advances in the application of kinetic isotope effects makes it possible to understand transition states for the complex interactions of RNA processing enzymes. Ricin-AB is a heterodimeric plant toxin, abundant in the castor bean. Subunit B is a galactose-specific lectin that provides cell entry to the complex followed by release of the A-chain, an adenine N-ribohydrolase specific for a single site on 28S rRNA. A single molecule of ricin is lethal for a mammalian cell, and a few ug are lethal for a human, making it among the most powerful cytotoxins. The adenine substrate resides in a hairpin stem-loop region of RNA, terminating in a GAGA tetraloop. The reaction mechanism for ricin A-chain forms a fully dissociated and transient ribooxacarbenium ion with closely related transition states. This knowledge has been used to produce the first generation of transition state analogue inhibitors for ricin A-chain, and is the most powerful catalytic site inhibitors known for the toxin. Second-generation transition state analogues for ricin a-chain will be designed from knowledge of the transition state structure. Investigation of the chemical mechanism of catalysis will use substrate specificity and site-directed mutagenesis studies. Structural analysis of ricin A-chain in complex with substrate, transition state and product analogues of RNA is intended to provide information on reaction coordinate motion and the role of individual amino acids in stabilizing the transition state complex. Powerful transition state inhibitors and chromogenic substrates for detecting ricin A-chain catalytic activity are two anticipated products of the research. These agents may be of use as rescue agents in immunochemotherapy and in detection of the toxin. The studies of ricin A-chain are intended to provide more complete knowledge of the reaction mechanisms for enzymes that recognize and covalently modify RNA. Consistent with this goal, studies will be initiated toward the transition state structure of an RNA site-specific adenylate deaminase, ADAR. Deamination at an adenylate site in mRNA produces an inosine site that is translated as a G, therefore causing A > G codon changes. Transition state inhibitors for these enzymes are anticipated to find use in altering protein expression and viral infectivity.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA072444-06
Application #
6543505
Study Section
Biochemistry Study Section (BIO)
Program Officer
Lees, Robert G
Project Start
1997-09-15
Project End
2007-06-30
Budget Start
2002-08-13
Budget End
2003-06-30
Support Year
6
Fiscal Year
2002
Total Cost
$377,582
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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Yuan, Hongling; Du, Quan; Sturm, Matthew B et al. (2015) Soapwort Saporin L3 Expression in Yeast, Mutagenesis, and RNA Substrate Specificity. Biochemistry 54:4565-74
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Sturm, Matthew B; Schramm, Vern L (2009) Detecting ricin: sensitive luminescent assay for ricin A-chain ribosome depurination kinetics. Anal Chem 81:2847-53
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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