Ricin A-chain: Kinetic isotope effects and transition state structure withstem-loop RNA

Ricin toxin A-chain (RTA) depurinates 28 S ribosomal RNA and small stem-loo p RNAs at the first adenosine residue in a 5'-GAGA-3' tetraloop. The transi tion state for depurination of stem-loop RNA by RTA was determined from kin etic isotope effects (KIEs). A stem-loop RNA, called A-10 (5'-GGCGAGAGCC-3' ), was synthesized using isotopically labeled ATP. KIEs were measured for R NA substrates with adenylates containing [1'-C-14], [9-N-15], [1'-C-14,9-N- 15], [7-N-15], [1'-H-3], [2'-H-3], [4'-H-3], or [5'-H-3]. Substrate-trappin g experiments established that the Michaelis complex of RTA.[C-14]A-10 diss ociates to free enzyme and [C-14]A-10 at least 20 times more frequently tha n its conversion to products, establishing minimal forward commitment to ca talysis. KIEs were used to interpret the transition-state structure. The ex perimental KIEs differ from previous N-ribohydrolase chemistries. Large KIE s were measured for [1'-H-3] (1.163 +/- 0.009) and [7-N-15] (0.981 +/- 0.00 8). A modest isotope effect occurred with [9-N-15] (1.016 +/- 0.005), and s mall KIEs were observed with [1'-C-14] (0.993 +/- 0.004) and [2'-H-3] (1.01 2 +/- 0.005). The experimental KIEs were analyzed using bond vibrational an d quantum chemical approaches, which demonstrated that a complex is formed of RTA with the RNA ribooxocarbenium ion and adenine that is in equilibrium with the Michaelis complex. A slow, irreversible, and nonchemical step is followed by nucleophilic attack by water. Release of the depurinated A-10 a nd adenine products is rapid. Other N-ribohydrolases catalyze dissociative concerted A(N)D(N) (S(N)2) transition states with weak participation of the leaving group and nucleophile. The KIEs for RTA establish a stepwise D-N*A (N) mechanism and the existence of a cationic intermediate with a finite Li fetime. The conformation of the ribosyl ring in the enzyme-stabilized RNA r ibooxocarbenium ion is 3'-endo, with an unusual dihedral angle of approxima tely 50 degrees between C2'-H2' and the vacant p-orbital of atom C1'. This conformation, which is unprecedented in N-ribohydrolases, is consistent wit h the geometry imposed by the stem-loop RNA backbone. These results establi sh that transition state analysis based on KIEs can be extended to the reac tions of nucleic acid chemistry.