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.
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