Abstract
Ricin toxin A-chain (RTA) depurinates 28 S ribosomal RNA and small stem-loop RNAs at the first adenosine residue in a 5‘-GAGA-3‘ tetraloop. The transition state for depurination of stem-loop RNA by RTA was determined from kinetic isotope effects (KIEs). A stem-loop RNA, called A-10 (5‘-GGCGAGAGCC-3‘), was synthesized using isotopically labeled ATP. KIEs were measured for RNA substrates with adenylates containing [1‘-14C], [9-15N], [1‘-14C,9-15N], [7-15N], [1‘-3H], [2‘-3H], [4‘-3H], or [5‘-3H]. Substrate-trapping experiments established that the Michaelis complex of RTA·[14C]A-10 dissociates to free enzyme and [14C]A-10 at least 20 times more frequently than its conversion to products, establishing minimal forward commitment to catalysis. KIEs were used to interpret the transition-state structure. The experimental KIEs differ from previous N-ribohydrolase chemistries. Large KIEs were measured for [1‘-3H] (1.163 ± 0.009) and [7-15N] (0.981 ± 0.008). A modest isotope effect occurred with [9-15N] (1.016 ± 0.0...
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