The transition state for RNA depurination by ricin a-chain

Ricin A-chain (RTA) catalyzes a specific depurination of ribosomal RNA at the elongation factor binding site, causing loss of protein biosynthesis. In vitro, RTA also catalyzes the hydrolysis of synthetic oligonucleotides. The Km and kcat for a 10-base stem-loop RNA, CGC GAGA GCG (A-10) are 4.1 #M and 4.1 rain-l, respectively. The purpose of this study is to characterize the transition state of RTA depurination by multiple V/K kinetic isotope effects (KIEs) using labeled A-10 as the substrate. The specifically labeled A-10 was synthesized enzymatically by conversion of labeled glucose or ribose 5'-phosphate to ATP and subsequently incorporation into RNA by RNA polymerase. KIEs for the hydrolysis are the following: primary 14C = 0.997 + 0.002, primary lSN = 1.020 :t: 0.004, a-secondary 3H = 1.163 + 0.010, /3-secondary aH= 1.011 :t: 0.006, -y-secondary 3H = 0.992 + 0.004, 6-secondary 3H = 0.996 4- 0.003, and primary double = 1.016 + 0.002. The KIEs are near-intrinsic since the commitment to catalysis is low. The results suggest that significant oxocarbenium character is developed in the transition state of depurination catalyzed by RTA.