A hypothesis to explain the substrate reactivity of ribosomal and stem-loop RNA with ricin A-chain.
A hypothesis is proposed which explains the low catalytic efficiency of ricin A-chain on artifical stem-loop RNA substrates, relative to the high catalytic efficiency found on intact mammalian ribosomes. The enzymatic binding energy required to reach the transition state is greater than that to stabilize the stem structure of stem-loop RNA molecules. When artifical stem-loop complexes bind, the base-pairing of the stem is lost rapidly relative to catalysis. Loss of secondary structure causes movement of the susceptible adenine to a catalytically unfavorable geometry and most of the enzyme-substrate complexes dissociate without catalysis. The protein architecture of intact ribosomes, when bound to ricin A-chain, is proposed to stabilize the stem-loop structure to maintain adenine 4324 in a configuration external to the RNA phosphodiester backbone. The slow catalysis observed with small stem-loop structures is a consequence of the relative probabilities for stem-melting induced by the enzyme and for reaching the transition state. Catalysis with a ten-base stem-loop RNA shows product formation at a rate of up to 0.02 hr-1, but fails to achieve a full catalytic turnover with long incubations. The substoichiometry of product formation with small stem-loops is proposed to be a consequence of the competing rates of catalysis and enzyme denaturation under in vitro assay conditions. Rates for these processes are estimated from the kinetic parameters for ricin A-chain hydrolysis of ribosomes and stem-loop structures.
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Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA.
Chen
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Niu
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D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation / Q000187:drug effects*; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012276:Ricin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D012276:Ricin