Transition State Structure of RNA Depurination by Saporin L3

Abstract

Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem–loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem–loop RNA (5′-GGGAGGGCCC-3′) by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.

Oligonucleotide transition state analogues of saporin L3

“…2) [22] while the MEP of the catalytically inactive substrate analogue PD p A p GPD differs significantly, which is also consistent with the weaker binding of this compound ( K i * = 16.1 μM; Fig. 2 and Table 1).…”

“…Saporin L3 and saporin L3 A14C were expressed as excreted proteins in yeast cultures and purified to near-homogeneity as previously reported [5,22]. …”

“…(a) Compound 2 (PD p DIA p G p PD, $K_{\mathrm{i}}^{\ast }$ 3.3 nM, from Yuan et al [22]. ); (b) compound 45 (PD p DEIA p G p pd, $K_{\mathrm{i}}^{\ast }$ 12.4 nM); (c) compound 54 (PD p HomoSer p G p PD, $K_{\mathrm{i}}^{\ast }$ 26.8 nM); and (d) catalytically inactive substrate analogue PD p A p G p PD ( $K_{\mathrm{i}}^{\ast }$ 16.1 μM).…”

Enzymatic Transition States and Drug Design

“…154 The intrinsic primary 1′– 14 C KIE for saporin L3 depurination is 1.052, less than expected for a fully developed S N 2 mechanism, where the 1′– 14 C KIE value can be as large as 1.14. 175–177 The smaller α-secondary hydrogen KIEs of 1.045 for saporin L3 compared to 1.163 for ricin A-chain also indicates a transition state with less dissociative character. Where does the bond order reside in the saporin L3 transition state?…”

Structural basis for neutralization of cytotoxic abrin by monoclonal antibody D6F10

“…Structure‐function studies have identified five invariant active site residues conserved among wide range of RIPs and have shed light on their possible roles in the depurination of A 4324 /A 2660 . The current accepted N‐glycosidase mechanism involves aromatic stacking of the adenine to be cleaved between two invariant tyrosines, its protonation by an invariant arginine residue, stabilization of the resulting ribose oxocarbenium (ribocation) like transition state by adjacent invariant glutamate residue and nucleophilic attack by a catalytic water molecule at the C1′ position of ribose carbocation intermediate resulting in the cleavage of N9‐C1′ N‐glycosidic bond of susceptible adenosine.…”