Transition State Structure of RNA Depurination by Saporin L3

Hongling Yuan, Christopher F. Stratton, Vern L. Schramm

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1383-1390
Number of pages8
JournalACS Chemical Biology
Volume11
Issue number5
DOIs
StatePublished - May 20 2016

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RNA
Isotopes
Kinetics
Adenine
Type 1 Ribosome Inactivating Proteins
Bearings (structural)
Ribosome Inactivating Proteins
Ricin
Immunotoxins
Nucleophiles
Transferases
saporin
Catalytic Domain
Substitution reactions
Carbon
Catalysts
Geometry
Water
Neoplasms
Therapeutics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

Transition State Structure of RNA Depurination by Saporin L3. / Yuan, Hongling; Stratton, Christopher F.; Schramm, Vern L.

In: ACS Chemical Biology, Vol. 11, No. 5, 20.05.2016, p. 1383-1390.

Research output: Contribution to journalArticle

Yuan, Hongling ; Stratton, Christopher F. ; Schramm, Vern L. / Transition State Structure of RNA Depurination by Saporin L3. In: ACS Chemical Biology. 2016 ; Vol. 11, No. 5. pp. 1383-1390.
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