Intrinsic pKa values of 3′-N-α-l-aminoacyl-3′- aminodeoxyadenosines determined by pH dependent 1H NMR in H 2O

Article abstract of DOI:10.1039/c0cc05136e

3′-(α-l-Aminoacylamido)deoxyadenosines are ribosomal A-site binders and mimic the nascent peptide accepting 3′-terminus of aminoacyl transfer RNA. Their α-amino groups exhibit intrinsic basicities in bulk water that differ by up to 1.8 pK_a unit

Full text of DOI:10.1039/c0cc05136e

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 3290–3292
www.rsc.org/chemcomm
COMMUNICATION
Intrinsic pKa values of 30-N-a-L-aminoacyl-30-aminodeoxyadenosines
1
determined by pH dependent H NMR in H2Ow
Kollappillil S. Krishnakumar,a Benoıt Y. Michel,a Nhat Quang Nguyen-Trung,a
Bernard Fenetb and Peter Strazewski*a
Received 23rd November 2010, Accepted 17th January 2011
DOI: 10.1039/c0cc05136e
30-(a-L-Aminoacylamido)deoxyadenosines are ribosomal A-site
binders and mimic the nascent peptide accepting 30-terminus of
aminoacyl transfer RNA. Their a-amino groups exhibit intrinsic
basicities in bulk water that differ by up to 1.8 pKa units. Only
the neutral form of these nucleophiles can be active during
ribosomal peptidyl transfer catalysis.
107 to 1010-fold reaction rate accelerations by the ribosome.
Nearly absent or weakly positive PT activation entropies
contrast a negative non-enzymatic activation entropy and
validate a model according to which the ribosome exhibits
throughout the catalyzed reaction pathway firm control over
the hydration of the reaction centres and overall H-bonding.7
A crucial prerequisite for this understanding is the certainty
about the rate-determining step during ribosomal peptide chain
elongation at speeds and under conditions that are relevant
in vivo. For decades one has tried to identify the rate-limiting
step in the peptide elongation cycle: after a suggestion that
mRNA translocation might be the slowest step followed
evidence that A-site occupation prior to PT could be rate
limiting. Fluorescence relaxation data have been interpreted
to imply that substrate accommodation into the A site is
about equally slow for all cognate aa-tRNA and largely
pH-independent, and that this step generally determines the
overall peptide elongation rate.8 Conversely, the relative
populations of reactive neutral versus inactive protonated
nucleophiles (a-amino groups of aa-tRNA) are expected to vary
quite markedly at physiological pH, depending on the intrinsic
pKa of each nucleophile and on the ability of the ribosomal A-site
to shift these intrinsic basicities into a perhaps more favorable
apparent regime. Thus, if the chemistry of the PT were rate-
limiting, rather than the accommodation of aa-tRNA in the A
site, one should expect the rate of protein synthesis to be
manifestly dependent on pH close to the physiological range
6–8 and reliant on the pKa-modulating effect of the 20 different
amino acid side chains linked to their A-site bound tRNAs.
Very recent pH dependent ribosomal PT experiments using
a fast in vitro translation assay unequivocally showed the
chemical step to be dominating the transfer rate of formyl
methionine from P-site bound fMet-tRNA onto A-site bound
prolyl- and glycyl-tRNA at or below physiological pH.9 MD
simulations on atomic coordinates of the large ribosomal
subunit containing a P-site bound dipeptidyl-tRNA and each
of the experimentally tested Asn-, Phe-, Ile-, Ala-, Gly- and
Pro-tRNA were carried out to calculate the pKa shifts of their
a-amino groups as they are being bound into the A-site in the
reactant state. For comparison, the experimental pKa shifts
were estimated from the differences between the pKa values at
25 1C of the a-amino groups of the corresponding natural
amino acids, taken from the literature10a and downshifted by
The ribosome catalyzes the transfer of nascent peptide chains
from 30-O-esters of P-site bound tRNAs to a-amino groups of
A-site bound 30-aminoacyl-tRNAs. These nucleophilic
a-amino groups are shown here to vary considerably in basicity
and thus in propensity to be in their neutral reactive form.
In Escherichia coli one codon nucleotide triplet is translated into
a new peptide bond during an average elongation time of 45 ms at
37 1C.1 This corresponds to an average protein elongation rate of
22 amino acids per second and ribosome. High in vitro elongation
rates1b are a prerequisite for peptidyl transfer (PT), rather than
foregoing aminoacyl-tRNA binding or accommodation steps,2 to
be kinetically observable. Much effort has been spent in the past
decade in studies of the molecular details of ribosomal PT through
modifications in the peptide-donating P site and its substrates.3
Peptide-accepting A-site substrates have also been studied. Here
knowledge of the basicity and nucleophilicity of the a-amino
group of incoming cognate 30-aminoacyl tRNA (aa-tRNA) is
of decisive importance.4 In vitro translation assays, in which
aa-tRNA or puromycin (1) were used as nascent peptide
acceptors, have been carried out at different temperatures and thus
elucidated a range of free energies of activation for the ribosome
a
catalyzed PT reaction: DGcat (25 1C) 16.5–12.6 kcal molꢀ1 1b,5
Carefully thought-out aqueous model reactions for the
.
a
bimolecular ‘uncatalyzed’ ester aminolysis revealed DGuncat
(25 1C) 22.2–23.5 kcal molꢀ1 5a,6
The difference translates into
.
a
b
´
Universite Lyon 1, CNRS UMR 5246, Institut de Chimie et
Biochimie Moleculaires et Supramoleculaires, Laboratoire de
´
´
`
´
Synthese de Biomolecules, 43 boulevard du 11 novembre 1918,
69622 Villeurbanne, France. E-mail: strazewski@univ-lyon1.fr
´
ˆ
CCRMN, Universite Lyon 1, CPE-Lyon, Bat. Curien,
43 bd du 11 novembre 1918, F-69622 Villeurbanne, France.
E-mail: bfenet@univ-lyon1.fr
w Electronic supplementary information (ESI) available: Synthetic
procedures, NMR data collection, fitting equations and parameters,
tables, literature data, SPARC analyses, fitted pH profiles, NMR
spectra for titration and compound characterisation. See DOI:
10.1039/c0cc05136e
c
3290 Chem. Commun., 2011, 47, 3290–3292
This journal is The Royal Society of Chemistry 2011

View Article Online
2.0 pKa units, and the apparent pKa values, defined as the pH at
which the rate of PT was half maximal at 20 1C.9 The remarkable
correlation suggests that under physiological conditions PT
‘chemistry’ could, in fact, be rate-limiting for all aa-tRNAs.
The lack of experimental intrinsic (bulk water) pKa values
for aa-tRNA is owed to the hydrolytic instability of the
adenosine-30-O-ester linkage making it impossible to obtain
reliable pKa data for 30-aminoacyl adenosine esters. Even
though a-amino acids and corrections based on experimental
pKa data of simple a-aminoacid methyl or ethyl esters11 may
indeed be good enough models to convey intrinsic pKa values
for a-amino groups of the natural A-site substrates
(aa-tRNA), we felt that the current efforts to ultimately under-
stand the ribosomal catalysis of cellular protein synthesis do call
for the best possible reference system. We thus decided to
experimentally determine pKa values for the a-amino groups
of true nascent peptide acceptors (A-site binders), viz. analogues
of puromycin that contain various natural or unnatural
L-amino acid side chains. In addition, we wished to put into
context our data with all available comparable experi-
mental pKa data of a-amino groups, and with those being
estimated by means of a state-of-the-art online calculator
named SPARC.12
Fig. 1 Puromycin analogues 1–8.
and alanine analogues 5 and 6, respectively. Here we report on
pKa values of the a-amino group of 1–6 in water. Analogues 7
and 8 served as controls to unmistakably distinguish the effect
of a-NH2 protonation from OH or CONH deprotonation.
Asn-, FPro-, Gly-, Ala-, Pro- and L-phenyllactic amides 2–6
and 8 are new puromycin analogues that were synthesized
according to published procedures16 and an optimised proto-
col for the purification and isolation of the very polar
target compounds (ESIw). 7 was obtained from 30-azido-30-
deoxyadenosine16 and Me2SO4 in aqueous KOH,17 followed
by standard reduction, coupling, deprotection and separation
protocols.wz
13C and 1H NMR spectroscopic analyses of aminodeoxy-
nucleosides gave pKa 6.2 and 6.9 for 20-NH2 and 30-NH2,
respectively.13a,b,d Most notably, pKa 6.2 was obtained for
20-NH2 in the absence or presence of a vicinal 30-phosphate
group (in dinucleotides). The a-amino group of puromycin,
being the most frequently used nascent peptide acceptor
analogue, was in the past first merely reported,13c then deter-
mined from a meticulous 500 MHz 1H NMR spectroscopic
study (5 mM in phosphate-buffered D2O), to exhibit pKa 7.3 at
25 1C.13d Later puromycin was titrated potentiometrically to
confer pKa 6.9 at 37 1C4a (cf. Table S3, ESIw) and pKa 7.2 at
25 1C4d (both 10 mM initial conc. in H2O). Analogues of
puromycin4d,f,g and other a-aminoacid amides14 were potentio-
metrically titrated in aqueous solutions that contained no,14a
small14b or Z 50% amounts of organic cosolvents. An
For the titration experiments, we avoided any buffer that
may interfere with the protonation of a-amino groups, be it
through solute complexation or self-dissociation and drastic
variation in ionic strength at pH 6–8.13d HCl, NaOH, and each
analyte at an initial concentration of 1.5 mM were dissolved in
H2O/D2O 9 : 1–95 : 5 that contained 0.15 M NaNO3, to
optimally buffer ionic strength18 being kept close to physio-
logical values throughout the titration: I = 0.15–0.18 M. All
1
pH-dependent H NMR spectra were taken at thermostated
298 K and 600 MHz (1 and 7)z or 500 MHz (2–6 and 8).y The
pH profiles of the amino acid side chain and anomeric 1H
NMR signals of puromycin (1) and analogues 2–8 showed that
signal shifts at pH 5.5–10 are absent only in 8, are thus due to
the ionization of a-NH2 (Fig. S3, Table S2, ESIw). H10 signal
shifts at pH o 4.5 (in 1–8) and pH 4 11 (in all but 7) attest
ionizations of adenine and, respectively, OH not CONH
(Fig. S2, Table S1, ESIw).
1
advantage of H NMR spectroscopy over potentiometric pH
titrations is the usually lower minimal amount and concentra-
tion of the analyte. Puromycin self-associates at Z 5 mM in
water.13e Both p-stacking and organic cosolvents are likely to
disfavor ionization and may diminish the apparent pKa value of
the a-amino group. NMR data taken in H2O/D2O Z 9 : 1, not
pure D2O, produce pKa values that are comparable without
pD–pH correction15 to pKa values that have been derived from
kinetic assays on ribosomes in H2O at pH 6–8.9
In the following list, the results are summarized in columns
A (compound no. and side chain) and B (experimental mean of
intrinsic pKa values at 25 1C for the a-amino groups ꢁ
0.01–0.07, cf. Table S2, ESIw). They are compared to the
predicted pKa values of the corresponding 30-deoxyadenosine-
30-a-aminoacyl amides (C: same compounds as in A) and the
corresponding 30-esters (D), as calculated by SPARC v4.5 for
25 1C (ibmlc2.chem.uga.edu/sparc). Experimental pKa values
from potentiometric titrations at 25 1C of simple a-aminoacyl
amides (in 0.15 M NaCl)14a and methyl esters (in 0.1 M KCl)11a
Given the importance of understanding the effect of all
twenty incoming natural peptide acceptors on PT catalysis,
and most likely on the overall rate of ribosomal protein
synthesis, we decided to investigate the full range of intrinsic
a-amino group basicities that a ribosomal A site usually has to
cope with. The least basic a-amino group of all proteinogenic
amino acids is that of L-asparagine (as in 2, Fig. 1); the most
basic a-amino group is that of L-proline (as in 3). We
wondered how strong the pKa-diminishing effect on an
L-proline derivative would be in which a g-H-atom of the side
chain had been replaced by a virtually isosteric F-atom
(as in 4), and what the precise pKa values are in the glycine-
are shown10b in columns
E and F, respectively. They
demonstrate that the replacement of a-CONH2 with
a-COOCH3 reduces the a-amino group’s basicity by
0.18–0.45 pKa units (0.48 and 0.51 for Tyr and Val). The
median difference is 0.31 pKa units (cf. Table S3, ESIw) and
reflects the electronegativity difference for 30-O vs. 30-N.
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 3290–3292 3291

View Article Online
(e) M. M. Changalov, G. D. Ivanova, M. A. Rangelov, P. Acharya,
S. Acharya, N. Minakawa, A. Foeldesi, I. B. Stoineva,
V. M. Yomtova, C. D. Roussev, A. Matsuda, J. Chattopadhyaya
and D. D. Petkov, ChemBioChem, 2005, 6, 992; (f) T. M. Schmeing,
K. S. Huang, D. E. Kitchen, S. A. Strobel and T. A. Steitz, Mol. Cell,
2005, 20, 437; (g) T. M. Schmeing, K. S. Huang, S. A. Strobel and
T. A. Steitz, Nature, 2005, 438, 520; (h) M. D. Erlacher, K. Lang,
B. Wotzel, R. Rieder, R. Micura and N. Polacek, J. Am. Chem. Soc.,
2006, 128, 4453; (i) J. S. Weinger and S. A. Strobel, Biochemistry,
2006, 45, 5939; (j) M. V. Rodnina, M. Beringer and W. Wintermeyer,
Trends Biochem. Sci., 2007, 32, 20; (k) M. Erlacher, D. N. Wilson,
R. Micura and N. Polacek, Chem. Biol., 2008, 15, 485; (l) M. Koch,
Y. Huang and M. Sprinzl, Angew. Chem., Int. Ed., 2008, 47, 7242.
4 (a) V. I. Katunin, G. W. Muth, S. A. Strobel, W. Wintermeyer and
M. V. Rodnina, Mol. Cell, 2002, 10, 339; (b) R. Green and
J. R. Lorsch, Cell, 2002, 110, 665; (c) A. C. Seila, K. Okuda,
For 1–6, both, pKa values and differences are somewhat
underestimated by SPARC—most pronounced for Asn, Ala
and Gly (not Pro) 30-amides—which predicts a corresponding
median amide vs. ester difference of 0.19 pKa units. Column G
shows the accordingly derived (B ꢀ 0.31) intrinsic pKa values
for 30-esters at 20 1C (same compounds as D); cooling by 5 1C
means to increase pKa by 0.10–0.15 units.9,11 The apparent
a-amino pKa values of A-site bound aa-tRNA at 20 1C
(ꢁ0.04–0.2) are reproduced from ref. 9 in H.
S. Nu´ nez, A. F. Seila and S. A. Strobel, Biochemistry, 2005, 44,
4018; (d) K. Okuda, A. C. Seila and S. A. Strobel, Biochemistry,
2005, 44, 6675; (e) B. Zhang, Z. Tan, L. Gartenmann Dickson,
M. N. L. Nalam, V. W. Cornish and A. C. Forster, J. Am. Chem.
Soc., 2007, 129, 11316; (f) D. A. Kingery, E. Pfund, R. M. Voorhees,
K. Okuda, I. Wohlgemuth, D. E. Kitchen, M. V. Rodnina and
S. A. Strobel, Chem. Biol., 2008, 15, 493; (g) K. Okuda, T. Hirota,
D. A. Kingery and H. Nagasawa, J. Org. Chem., 2009, 74, 2609.
5 (a) A. Sievers, M. Beringer, M. V. Rodina and R. Wolfenden,
Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 7897; (b) M. Beringer,
C. Bruell, L. Xiong, P. Pfister, P. Bieling, V. I. Katunin,
The apparent basicities of aa-tRNA’s nucleophilic a-amino
groups that are reacting in the ribosome’s A site can differ by up
to 1.9 pKa units (for Asn- vs. Pro-tRNA).9 Given equimolar
total concentrations and a local pH 7.5, the nucleophiles of
a-amino-Asn : Pro thus appear neutral in a 3 : 1 molar ratio
([1 + 107.8ꢀpH]/[1 + 105.9ꢀpH], H) (ESIw). This study shows that,
within r0.2 pKa limits, the intrinsic basicities of the same
a-amino groups at 20 1C (G) compare well with earlier less
directly derived values9 and span about the same 1.8 pKa (B) as
that of A site-bound aa-tRNA. Molar ratios for neutral
(equimolar total) Asn : Pro a-amines in bulk water at pH 7.5
are 10 : 1 for 30-amides 2 : 3 (Fig. S1, ESIw) and 7–8 : 1 for
30-esters. FPro 4 sterically stands in for Pro 3 but its a-NH is as
weakly basic as that of Asn 2.
A. S. Mankin, E. C. Bottger and M. V. Rodnina, J. Biol. Chem.,
¨
2005, 280, 36065; (c) I. Wohlgemuth, S. Brenner, M. Beringer and
M. V. Rodnina, J. Biol. Chem., 2008, 283, 32229.
6 (a) A. Sievers, M. Beringer, M. V. Rodina and R. Wolfenden, Proc.
Natl. Acad. Sci. U. S. A., 2004, 101, 12397; (b) G. K. Schroeder and
R. Wolfenden, Biochemistry, 2007, 46, 4037.
7 (a) P. K. Sharma, Y. Xiang, M. Kato and A. Warshel, Biochemistry,
2005, 44, 11307; (b) S. Trobro and J. Aqvist, Proc. Natl. Acad. Sci.
U. S. A., 2005, 102, 12395; (c) S. Trobro and J. Aqvist, Biochemistry,
2006, 45, 7049; (d) A. Gindulyte, A. Bashan, I. Agmon, L. Massa,
A. Yonath and J. Karle, Proc. Natl. Acad. Sci. U. S. A., 2006, 103,
13327; (e) G. Wallin and J. Aqvist, Proc. Natl. Acad. Sci. U. S. A.,
2010, 107, 1888.
8 (a) M. V. Rodnina, R. Fricke and W. Wintermeyer, Biochemistry,
1994, 33, 12267; (b) T. Pape, W. Wintermeyer and M. V. Rodnina,
EMBO J., 1998, 17, 7490.
9 M. Johansson, K.-W. Ieong, S. Trobro, P. Strazewski, J. Aqvist,
M. Y. Pavlov and M. Ehrenberg, Proc. Natl. Acad. Sci. U. S. A.,
2010, 108, 79.
We thank Mans Ehrenberg and Anthony Foster for helpful
discussions. Financial support was provided by FP6 programme
Synthcells and COST Action CM0703 Systems Chemistry.
10 (a) A. Fersht, Structure and Mechanism in Protein Science: A Guide to
Enzyme Catalysis and Protein Folding, Freeman & Co, New York,
1999; (b) H. A. Sober, CRC Handbook of Biochemistry, 2nd edn, 1970.
11 (a) R. W. Hay and L. J. Porter, J. Chem. Soc. B, 1967, 1261;
(b) R. W. Hay and P. J. Morris, J. Chem. Soc. B, 1970, 1577.
12 (a) S. H. Hilal, S. W. Karickhoff and L. A. Carreira, Quant.
Struct.–Act. Relat., 1995, 14, 348; (b) A. C. Lee and G. M.
Crippen, J. Chem. Inf. Model., 2009, 49, 2013; (c) D. D. Perrin,
B. Dempsey and E. P. Serjeant, pKa Prediction for Organic Acids
and Bases, Chapman & Hall, New York, 1981.
13 (a) P. S. Miller, B. Purshotam and K. Lou-Sing, Nucleosides,
Nucleotides Nucleic Acids, 1993, 12, 785; (b) H. Aurup,
T. Tuschl, F. Benseler, J. Ludwig and F. Eckstein, Nucleic Acids
Res., 1994, 22, 20; (c) G. M. Cathey and S. J. Klebanoff, Biochim.
Biophys. Acta, 1967, 145, 806; (d) S. S. Narula and M. M. Dhingra,
Indian J. Biochem. Biophys., 1986, 23, 306; (e) S. S. Narula and
M. M. Dhingra, J. Biomol. Struct. Dyn., 1984, 2, 191.
Notes and references
z The synthesis and pH-dependent 1H NMR spectroscopic analysis of
1 and 7 were part of the PhD thesis of N. Q. Nguyen-Trung, 2003,
University of Basel, Switzerland.
y Prior to fitting and plotting, the to be analysed chemical shifts dH of
all compounds 1–8 were corrected for the pH dependence of the
internal standard (CH3)3SiCD2CD2COONa (TSP): dH(TSP)
ꢂ
0.00 ppm, pKa (TSP) = 5.00, via the Henderson–Hasselbalch equation
dH(pH) = 2.217 + {ꢀ2.217 ꢀ 2.236ꢃ10pHꢀ5.00/(1 + 10pHꢀ5.00)}.19 All
corrected datapoints dHa, dHb, dHg, dHd furnished pH profiles that are
pHꢀpKa
best fits for dH(pH) = dA + (dB ꢀ dA)ꢃ10pHꢀpK /(1 + 10
),
a
where the base lines (dA, dB) and the transition midpoint (pKa) served
as free fitting parameters.w
1 (a) M. Y. Pavlov, R. E. Watts, Z. Tan, V. W. Cornish,
M. Ehrenberg and A. C. Forster, Proc. Natl. Acad. Sci. U. S. A.,
2008, 106, 50; (b) M. Johansson, E. Bouakaz, M. Lovmar and
M. Ehrenberg, Mol. Cell, 2008, 30, 589.
2 S. Ledoux and O. C. Uhlenbeck, Mol. Cell, 2008, 31, 114.
3 (a) G. K. Das, D. Bhattacharyya and D. P. Burma, J. Theor. Biol.,
1999, 200, 193; (b) S. Dorner, C. Panuschka, W. Schmid and
A. Barta, Nucleic Acids Res., 2003, 31, 6536; (c) J. S. Weinger,
K. M. Parnell, S. Dorner, R. Green and S. A. Strobel, Nat. Struct.
Mol. Biol., 2004, 11, 1101; (d) M. D. Erlacher, K. Lang,
14 (a) R. W. Chambers and F. H. Carpenter, J. Am. Chem. Soc., 1955, 77,
1523; (b) R. E. Watts and A. C. Forster, Biochemistry, 2010, 49, 2177.
15 A. Kre˛zel and W. Bal, J. Inorg. Biochem., 2004, 98, 161.
˙
16 (a) H. Chapuis and P. Strazewski, Tetrahedron, 2006, 62, 12108;
(b) A. Charafeddine, W. Dayoub, H. Chapuis and P. Strazewski,
Chem.–Eur. J., 2007, 13, 5566; (c) B. Y. Michel, K. S.
Krishnakumar and P. Strazewski, Synlett, 2008, 246; (d) B. Y.
Michel and P. Strazewski, Chem.–Eur. J., 2009, 15, 6244.
17 J. T. Kazimierczuk, E. Darzynkiewicz and D. Shugar,
Biochemistry, 1976, 15, 2735.
N. Shankaran, B. Wotzel, A. Huttenhofer, R. Micura,
¨
A. S. Mankin and N. Polacek, Nucleic Acids Res., 2005, 33, 1618;
18 R. Sigel and H. Sigel, Acc. Chem. Res., 2010, 43, 974.
19 A. De Marco, J. Magn. Reson., 1977, 26, 527.
c
3292 Chem. Commun., 2011, 47, 3290–3292
This journal is The Royal Society of Chemistry 2011