A R T I C L E S
Luo and Schramm
Figure 2. TadA-mediated adenosine deamination. The initial step proposed
for TadA-catalyzed demination is formation of Zn2+-hydroxyl. The nearby
Glu residue serves as the general base to remove proton from Zn-bound
water. The structures of guanosine and the product inosine are shown.
Inosine in edited RNA is recognized as guanosine.
Figure 3. Measurement of forward commitment factor of ecTadA.18,19 The
isotope trapping experiment for measuring the forward commitment was
performed under rapid-mixing pre-steady-state conditions. Enzyme was
bound to the labeled substrate in a 2 ms mix and was then diluted into a
large excess of unlabeled cold substrate. The ratio of IMP formed to enzyme-
bound substrate was plotted vs time. The forward commitment factor (Cf)
was calculated from the ordinate intercept, fit to a linear equation with Cf
) 0.15.
Recent studies on human ADAR substrates further led to
identifying a new A-to-I editing target BC10 that has been linked
to bladder cancer and renal cancer proliferation. hADAR is also
characterized as one of few unambiguously up-regulated genes
in solid tumors and liver cancer.12-14 These pieces of evidence
strongly relate ADAR upregulation to cancer progression.
ADARs also process microRNAs and therefore alter expression
or target specificity of the microRNAs.15,16 Our interest in
transition-state structure led us to solve the transition state
structure of TadA, an RNA editing enzyme which targets a
structurally complex and biologically relevant tRNAs from
Escherichia coli (Figures 1 and 2).
KIEs are one of the few tools to characterize enzyme
transition states in structural detail.17-20 The elucidation of
enzyme transition state structure is valuable in the development
of potent inhibitors. Molecules that mimic enzymatic transition
states are expected to show Kd values of 10-18-10-21 M,
proportional to the enzyme-imposed rate enhancement of
catalysis.21 We have applied this approach to achieve inhibitors
with pM to fM affinities for N-ribosyltransferase enzymes.21
Here we apply similar approaches to probe the more complex
transition state structure of TadA. The results are of potential
utility for the design of transition state analogue inhibitors in
the family of RNA editing enzymes.
1.014, 1.022, and 0.993 were obtained for primary [6-13C]-,
[6-15N], and secondary [1-15N] positions, respectively. This suite
of KIEs was used as constraints to model a transition state for
ecTadA. ecTadA adopts a late SNAr transition state with a
complete, pro-S-face hydroxyl addition, nearly complete N1
protonation and significant N6-C6 dissociation. The rate-
limiting step of the ecTadA-catalyzed deamination is subsequent
to the formation of the tetrahedral Meisenheimer intermediate.
Computational analysis of the reaction supports a transition state
with a proton shuttle between the hydroxyl nucleophile and the
leaving group N6, mediated by nearby Glu70. Significant
distortion of the 5′-sugar and rotation of the ribosidic purine
on the path to transition state formation were indicated from
the large inverse [5′-3H2] KIE of -4% and significant [1′-3H]
KIE of 1.4%. This is the first time that KIEs have been
correlated with purine base rotation at an RNA-based transition
state. We propose that this KIE approach will be useful for
transition state and base rotation characterization of other RNA
editing enzymes. The ecTadA transition state structure reveals
the detailed architecture for catalytic efficiency and provides a
blueprint for designing tight-binding inhibitors.
Kinetic isotope effects are reported for the deamination of
RNA by E. coli TadA using substrates whose editing sites were
labeled with [6-13C], [6-15N], [1-15N], [5′-3H2], and [1′-3H]. The
transition state structure of ecTadA was explored with KIE-
constrained quantum chemical calculations. Intrinsic KIEs of
Experimental Section
(8) Macbeth, M. R.; Schubert, H. L.; VanDemark, A. P.; Lingam, A. T.; Hill,
C. P.; Bass, B. L. Science 2005, 309, 1534-1539.
Expression and Purification of ecTadA. A 13aa-truncated ecTadA
was expressed and purified as described previously with some
modification.3 Here the ecTadA was subject to two additional MonoQ
columns (GE Science) and a Superdex75 purification (GE Science) to
lower RNase activities.
Synthesis of Isotopically Labeled Substrates. [1′-3H]-, [5′-3H2]-,
[1′-14C]-, [1′-14C, 6-13C], [1′-14C, 6-15N], [1′-14C, 6-13C15N], and [1′-
14C, 1-15N]-labeled ATPs were prepared enzymatically as described
previously.18,19 The isotopically labeled stem loops were synthesized
by incorporating labeled ATPs via the T7 RNA polymerase reaction
(MEGAshort script T7 kit, Ambion). See Supporting Information for
detailed information.
(9) Tonkin, L. A.; Saccomanno, L.; Morse, D. P.; Brodigan, T.; Krause, M.;
Bass, B. L. EMBO J. 2002, 21, 6025-6035.
(10) Wong, S. K.; Lazinski, D. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
15118-15123.
(11) Sato, S.; Wong, S. K.; Lazinski, D. W. J. Virol. 2001, 75, 8547.
(12) Pilarsky, C.; Wenzig, M.; Specht, T.; Saeger, H. D.; Grutzmann, R.
Neoplasia 2004, 6, 744-750.
(13) Midorikawa, Y.; Tsutsumi, S.; Taniguchi, H.; Ishii, M.; Kobune, Y.;
Kodama, T.; Makuuchi, M.; Aburatani, H. Jpn. J. Cancer Res. 2002, 93,
636-643.
(14) (a) Clutterbuck, D. R.; Leroy, A.; O’Connell, M. A.; Semple, C. A.
Bioinformatics 2005, 21, 2590-2595. (b) Gromova, I.; Gromov, P.; Celis,
J. E. Int. J. Cancer 2002, 98, 539-546.
(15) Kawahara, Y.; Zinshteyn, B.; Sethupathy, P.; Iizasa, H.; Hatzigeorgiou,
A. G.; Nishikura, K. Science 2007, 315, 1137-1140.
(16) Yang, W. D.; Chendrimada, T. P.; Wang, Q. D.; Higuchi, M.; Seeburg, P.
H.; Shiekhattar, R.; Nishikura, K. Nat. Struct. Mol. Biol. 2006, 13, 13-21.
(17) Cleland, W. W. Arch. Biochem. Biophys. 2005, 433, 2-12.
(18) Luo, M.; Singh, V.; Taylor, E. A.; Schramm, V. L. J. Am. Chem. Soc.
2007, 129, 8008-8017.
(19) Singh, V.; Schramm, V. L. J. Am. Chem. Soc. 2007, 129, 2783-2795.
(20) Schramm, V. L. Curr. Opin. Struct. Biol. 2005, 15, 604-613.
(21) Schramm, V. L. Arch. Biochem. Biophys. 2005, 433, 13-26.
Measurement of Commitment Factor. Forward commitment for
ecTadA-catalyzed deamination was determined by an isotope trapping
method under rapid-mixing presteady-state conditions (Figure 3).18,19
Briefly, 25.1 µL of 30 µM ecTadA stock was rapidly mixed with 20.9
µL of 36.5 µM [1′-3H]-labeled ectRNAarg2 stem loop substrate (total 2
× 105 cpm) for 2 ms using a quench flow apparatus (PQF-3, KinTek).
9
2650 J. AM. CHEM. SOC. VOL. 130, NO. 8, 2008