posted on 2016-02-17, 00:00authored byHongling Yuan, Christopher F. Stratton, Vern L. Schramm
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.