Transition state of ADP-ribosylation of acetyllysine catalyzed by archaeoglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches

Published on Web 08/18/2010

Transition State of ADP-Ribosylation of Acetyllysine

Catalyzed by Archaeoglobus fulgidus Sir2 Determined by

Kinetic Isotope Effects and Computational Approaches

Yana Cen and Anthony A. Sauve*

Department of Pharmacology, Weill Medical College of Cornell UniVersity, 1300 York AVenue,

New York, New York 10065

Received December 8, 2009; E-mail: aas2004@med.cornell.edu

Abstract: Sirtuins are protein-modifying enzymes distributed throughout all forms of life. These enzymes

bind NAD⁺, a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein

side chains. This NAD⁺-dependent deacetylation reaction has been observed for sirtuin enzymes derived

from archaeal, eubacterial, yeast, metazoan, and mammalian species, suggesting conserved chemical

mechanisms for these enzymes. The ﬁrst chemical step of deacetylation is the reaction of NAD⁺with an

acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide.

In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeoglobus

fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method

and were [1_N-¹⁵N] ) 1.024(2), [1′_N-¹⁴C] ) 1.014(4), [1′_N-³H] ) 1.300(3), [2′_N-³H] ) 1.099(5), [4′_N-³H] )

0.997(2), [5′_N-³H] ) 1.020(5), [4′_N-¹⁸O] ) 0.984(5). KIEs were calculated for candidate transition state

structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and

experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes

a highly dissociated oxocarbenium ionlike transition state with very low bond orders to the leaving group

nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism

forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.

mammalian sirtuins regulate adipogenesis,¹⁶adipolysis,¹⁶

apoptosis,^17-20insulin secretion,^21,22gluconeogenesis,^23,24mi-

tochondrial biogenesis,^25,26metabolic pathways,^25,26DNA

1. Introduction

The sirtuins are phylogenetically conserved NAD⁺dependent

enzymes that regulate diverse signaling processes within cells

via protein covalent modiﬁcation.^1,2The sirtuins catalyze

removal of protein acetyllysines, although in some cases they

have been reported to ADP-ribosylate proteins and other

substrates as well.^3-8The sirtuins have been implicated in

cellular and organism adaptations to stress and nutrient

intake,^2,9-11and regulate lifespan in different organisms includ-

ing yeast,¹²worms,¹³ﬂies¹⁴and probably in mammals.¹⁵The

(13) Tissenbaum, H. A.; Guarente, L. Nature 2001, 410, 227–230.

(14) Rogina, B.; Helfand, S. L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,

15998–16003.

(15) Dali-Youcef, N.; Lagouge, M.; Froelich, S.; Koehl, C.; Schoonjans,

K.; Auwerx, J. Ann. Med. 2007, 39, 335–345.

(16) Picard, F.; Kurtev, M.; Chung, N.; Topark-Ngarm, A.; Senawong, T.;

Machado De Oliveira, R.; Leid, M.; McBurney, M. W.; Guarente, L.

Nature 2004, 429, 771–776.

(17) Luo, J.; Nikolaev, A. Y.; Imai, S.; Chen, D.; Su, F.; Shiloh, A.;

Guarente, L.; Gu, W. Cell 2001, 107, 137–148.

(1) Sauve, A. A.; Wolberger, C.; Schramm, V. L.; Boeke, J. D. Annu.

ReV. Biochem. 2006, 75, 435–465.

(18) Vaziri, H.; Dessain, S. K.; Ng Eaton, E.; Imai, S. I.; Frye, R. A.;

Pandita, T. K.; Guarente, L.; Weinberg, R. A. Cell 2001, 107, 149–

159.

(2) Blander, G.; Guarente, L. Annu. ReV. Biochem. 2004, 73, 417–435.

(3) French, J. B.; Cen, Y.; Sauve, A. A. Biochemistry 2008, 47, 10227–

10239.

(19) Yamakuchi, M.; Ferlito, M.; Lowenstein, C. J. Proc. Natl. Acad. Sci.

U.S.A. 2008, 105, 13421–13426.

(4) Merrick, C. J.; Duraisingh, M. T. Eukaryotic Cell 2007, 6, 2081–

2091.

(20) Firestein, R.; Blander, G.; Michan, S.; Oberdoerffer, P.; Ogino, S.;

Campbell, J.; Bhimavarapu, A.; Luikenhuis, S.; de Cabo, R.; Fuchs,

C.; Hahn, W. C.; Guarente, L. P.; Sinclair, D. A. PLoS One 2008, 3,

e2020.

(5) Du, J.; Jiang, H.; Lin, H. Biochemistry 2009, 48, 2878–2890.

(6) Haigis, M. C.; Mostoslavsky, R.; Haigis, K. M.; Fahie, K.; Christodou-

lou, D. C.; Murphy, A. J.; Valenzuela, D. M.; Yancopoulos, G. D.;

Karow, M.; Blander, G.; Wolberger, C.; Prolla, T. A.; Weindruch,

R.; Alt, F. W.; Guarente, L. Cell 2006, 126, 941–954.

(7) Kowieski, T. M.; Lee, S.; Denu, J. M. J. Biol. Chem. 2008, 283, 5317–

5326.

(21) Moynihan, K. A.; Grimm, A. A.; Plueger, M. M.; Bernal-Mizrachi,

E.; Ford, E.; Cras-Meneur, C.; Permutt, M. A.; Imai, S. Cell Metab.

2005, 2, 105–117.

(22) Bordone, L.; Motta, M. C.; Picard, F.; Robinson, A.; Jhala, U. S.;

Apfeld, J.; McDonagh, T.; Lemieux, M.; McBurney, M.; Szilvasi, A.;

Easlon, E. J.; Lin, S. J.; Guarente, L. PLoS Biol. 2006, 4, e31.

(23) Liu, Y.; Dentin, R.; Chen, D.; Hedrick, S.; Ravnskjaer, K.; Schenk,

S.; Milne, J.; Meyers, D. J.; Cole, P.; Yates, J., III; Olefsky, J.;

Guarente, L.; Montminy, M. Nature 2008, 456, 269–273.

(24) Rodgers, J. T.; Lerin, C.; Haas, W.; Gygi, S. P.; Spiegelman, B. M.;

Puigserver, P. Nature 2005, 434, 113–118.

(8) Garcia-Salcedo, J. A.; Gijon, P.; Nolan, D. P.; Tebabi, P.; Pays, E.

EMBO J. 2003, 22, 5851–5862.

(9) Haigis, M. C.; Guarente, L. P. Genes DeV. 2006, 20, 2913–2921.

(10) Guarente, L.; Picard, F. Cell 2005, 120, 473–482.

(11) Yang, T.; Fu, M.; Pestell, R.; Sauve, A. A. Trends Endocrinol. Metab.

2006, 17, 186–191.

(12) Lin, S. J.; Defossez, P. A.; Guarente, L. Science 2000, 289, 2126–

2128.

(25) Rodgers, J. T.; Lerin, C.; Gerhart-Hines, Z.; Puigserver, P. FEBS Lett.

2008, 582, 46–53.

9

12286 J. AM. CHEM. SOC. 2010, 132, 12286–12298

10.1021/ja910342d  2010 American Chemical Society

Transition State of Sirtuins

A R T I C L E S

Scheme 1. Reaction Scheme for Af2Sir2-Catalyzed Deacetylation

repair,^20,27-30senescence,³⁰and proliferation.^20,31,32Sirtuins are

profound regulators of diverse biological processes and their

chemical and enzymatic mechanisms are of heightened interest.

In turn, the development of small molecule modulators of

sirtuins is an active area of research.^33-37

In addition to fascinating biological effects, the sirtuins have

unusual chemistry.¹The sirtuins deacetylate proteins using

NAD⁺as a cosubstrate and are sensitive to changes in cellular

NAD⁺and nicotinamide concentrations.^38-40Sirtuin catalyzed

deacetylation generates nicotinamide and the acetyl-transfer

product, 2′-O-acetyl-ADPribose (2′-AADPR).^41,42Sauve et al.

proposed that the ﬁrst step in sirtuin catalyzed deacetylation is

ADP-ribosyltransfer to the acetyllysine group of a protein or

peptide substrate to form an intermediate called an O-alkyla-

midate,⁴¹also called an ADP-ribosyl-peptidylimidate (Scheme

1).¹The intermediate has versatile properties. For example,

NAD⁺can be reformed from it when nicotinamide concentra-

tions are elevated in solution (Scheme 1).⁴¹Chemical reversal

accounts for the inhibition of sirtuins by metabolic and

pharmacologic nicotinamide concentrations.^39,40Alternatively,

the imidate reacts forward via nucleophilic attack of the 2′-OH

on the imidate, resulting in deacetylation and enzymatic

formation of 2′-AADPR (Scheme 1).⁴¹This mechanistic pro-

posal has explained a variety of chemistries of sirtuins reported

by numerous laboratories; however, direct characterizations of

mechanistic intermediates or transition states have not been

reported.

The ADP-ribosylation of the acetyllysine moiety was pro-

posed to involve electrophilic capture of the acetyl-oxygen by

an oxacarbenium ionlike transition state, characterized by low

bond orders to leaving group and nucleophile (Scheme 2 PATH

A).^41,43Signiﬁcant mechanistic precedent for this reaction was

available from determinations of transition states of both

enzymatic and nonenzymatic ADP-ribosylation reactions. For

example, kinetic isotope effect (KIE) measurements and com-

putational chemistry had elucidated well-formed oxacarbenium

ionlike transition states for ADP-ribosyltransfer reactions of the

NAD⁺-dependent bacterial toxins^44-47and hydrolysis of NAD⁺

in water (pH ) 5).⁴⁸Such an electrophilic mechanism overcomes

the limitation of the acetyllysine as a poor nucleophile. We

disfavored an S_N2-type mechanism, distinguished by signiﬁcant

association of the nucleophile at the transition state (Scheme 2,

PATH B). Crystallography and mutagenesis also indicated that a

proximate base at the active site for nucleophilic activation of the

amide was not necessary for the ADP-ribosylation.^1,49

(26) Gerhart-Hines, Z.; Rodgers, J. T.; Bare, O.; Lerin, C.; Kim, S. H.;

Mostoslavsky, R.; Alt, F. W.; Wu, Z.; Puigserver, P. EMBO J. 2007,

26, 1913–1923.

(27) Wang, R. H.; Sengupta, K.; Li, C.; Kim, H. S.; Cao, L.; Xiao, C.;

Kim, S.; Xu, X.; Zheng, Y.; Chilton, B.; Jia, R.; Zheng, Z. M.; Appella,

E.; Wang, X. W.; Ried, T.; Deng, C. X. Cancer Cell 2008, 14 (4),

312–323.

(28) O’Hagan, H. M.; Mohammad, H. P.; Baylin, S. B. PLoS Genet. 2008,

4, e1000155.

(29) Mostoslavsky, R. Front. Biosci. 2008, 13, 6966–6990.

(30) Chua, K. F.; Mostoslavsky, R.; Lombard, D. B.; Pang, W. W.; Saito,

S.; Franco, S.; Kaushal, D.; Cheng, H. L.; Fischer, M. R.; Stokes, N.;

Murphy, M. M.; Appella, E.; Alt, F. W. Cell Metab. 2005, 2, 67–76.

(31) Mantel, C.; Broxmeyer, H. E. Curr. Opin. Hematol. 2008, 15, 326–

331.

(32) Brooks, C. L.; Gu, W. Nat. ReV. Cancer 2009, 9, 123–128.

(33) Smith, B. C.; Hallows, W. C.; Denu, J. M. Chem. Biol. 2008, 15,

1002–1013.

Since the initial mechanistic proposal was published, debate

about the mechanism of the ADP-ribosylation step has increased.

(34) Jiang, W. J. Biochem. Biophys. Res. Commun. 2008, 373, 341–344.

(35) Milne, J. C.; et al. Nature 2007, 450, 712–716.

(36) Lara, E.; et al. Oncogene 2009, 28, 781–791.

(43) Sauve, A. A.; Schramm, V. L. Curr. Med. Chem. 2004, 11, 807–826.

(44) Scheuring, J.; Schramm, V. L. Biochemistry 1997, 36, 8215–8223.

(45) Scheuring, J.; Schramm, V. L. Biochemistry 1997, 36, 4526–4534.

(46) Berti, P. J.; Blanke, S. R.; Schramm, V. L. J. Am. Chem. Soc. 1997,

119, 12079–12088.

(37) Sauve, A. A. Curr. Pharm. Des. 2009, 15, 45–56.

(38) Denu, J. M. Trends Biochem. Sci. 2005, 30, 479–483.

(39) Jackson, M. D.; Schmidt, M. T.; Oppenheimer, N. J.; Denu, J. M.

J. Biol. Chem. 2003, 278, 50985–50998.

(47) Parikh, S. L.; Schramm, V. L. Biochemistry 2004, 43, 1204–1212.

(48) Berti, P. J.; Schramm, V. L. J. Am. Chem. Soc. 1997, 119, 12069–

12078.

(40) Sauve, A. A.; Schramm, V. L. Biochemistry 2003, 42, 9249–9256.

(41) Sauve, A. A.; Celic, I.; Avalos, J.; Deng, H.; Boeke, J. D.; Schramm,

V. L. Biochemistry 2001, 40, 15456–15463.

(49) Min, J.; Landry, J.; Sternglanz, R.; Xu, R. M. Cell 2001, 105, 269–

279.

(42) Jackson, M. D.; Denu, J. M. J. Biol. Chem. 2002, 277, 18535–18544.

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A R T I C L E S

Cen and Sauve

Scheme 2. Proposed Mechanisms for Af2Sir2-Catalyzed Deacetylation: (A) Oxacarbenium Ionlike, Dissociated Transition State; (B)

Non-oxacarbenium Ion, Nucleophile-Associated Transition State

A provocative study by Smith and Denu generated evidence

that the nucleophile has “considerable participation” in the ADP-

ribosylation step.⁵⁰Modiﬁed acetyllysine analogues, such as

R-monoﬂuoro, diﬂuoro, and triﬂuoro derivatives, provided

extremely slow rates of NAD⁺turnover, leading the authors to

conclude that “the rate of nicotinamide-ribosyl bond cleavage

[is] consistent with an S_N2-like mechanism”⁵⁰and inconsistent

with a fully dissociated transition state oxacarbenium ion.^41,50

More recently, both high-level computational QM/MM⁵¹and

X-ray crystallographic studies⁵²have supported a largely

dissociated transition state structure, although the computational

study concurred that the transition state had some association

of the nucleophile.⁵¹

The most rigorous experimental determination of transition

state structure is from KIEs, which have the advantage of

probing the transition state directly without perturbing substrate

electronics or sterics. We chose this approach to determine the

transition state for the ﬁrst chemical step of a sirtuin. The

transition state for the nicotinamide cleavage/ADP-ribosylation

step was solved by the method of matching experimental KIEs

to calculated KIEs for candidate transition state structures. These

methods have solved a large number of transition state structures

of N-ribosyltransferase enzymes,^44-47,53,54including thymidine

phosphorylase which proceeds via an associative mechanism.⁵⁵

In this study, we solve the transition state structure of an

Archaeoglobus fulgidus sirtuin, Af2Sir2. This enzyme had

previously been studied by X-ray crystallography⁵⁶and has well-

characterized deacetylase⁴¹and base-exchange reactions.⁴⁰We

report here the transition state structure of the ADP-ribosylation

step to acetyllysine for this enzyme and conclude that this sirtuin

stabilizes an oxacarbenium ionlike transition state with very low

bond orders to nicotinamide and acetyl oxygen.

2. Materials and Methods

General Materials. Archaeoglobus fulgidus Af2Sir2 was

expressed from a plasmid as previously described.⁴⁰A peptide

based on p53 sequence (HLKSKKGQSTSRHK(K-Ac)LMFK),

called p53A, was synthesized by the Proteomics Resource

Center at Rockefeller University. Enzymes and reagents for

NAD⁺synthesis are reported in the Supporting Information.

Synthesis of Isotopically Labeled Substrates. [1′_N-³H],

[4′_N-³H], [5′_N-³H], [1′_N-¹⁴C], [2,8_A-³H], [8_A-¹⁴C], and [1-¹⁵N-

5′_N-¹⁴C]-NAD⁺’s (99% 1-¹⁵N) were prepared enzymatically as

described previously.⁵⁷[4′_N-¹⁸O-2,8_A-³H]-NAD⁺(95% 4′_N-¹⁸O)

was synthesized by modiﬁcation of methods reported for making

isotopically labeled NAD⁺’s,^46,57and this method is included

in the Supporting Information. Detailed chemical and enzymatic

synthesis of [2′_N-³H]-NAD⁺is described in the Supporting

Information.

(50) Smith, B. C.; Denu, J. M. J. Am. Chem. Soc. 2007, 129, 5802–5803.

(51) Hu, P.; Wang, S.; Zhang, Y. J. Am. Chem. Soc. 2008, 130, 16721–

16728.

(55) Birck, M. R.; Schramm, V. L. J. Am. Chem. Soc. 2004, 126, 2447–

(52) Hawse, W. F.; Hoff, K. G.; Fatkins, D. G.; Daines, A.; Zubkova, O. V.;

Schramm, V. L.; Zheng, W.; Wolberger, C. Structure 2008, 16, 1368–

1377.

2453.

(56) Avalos, J. L.; Celic, I.; Muhammad, S.; Cosgrove, M. S.; Boeke, J. D.;

Wolberger, C. Mol. Cell 2002, 10, 523–535.

(53) Schramm, V. L. J. Biol. Chem. 2007, 282, 28297–28300.

(54) Schramm, V. L. Arch. Biochem. Biophys. 2005, 433, 13–26.

(57) Rising, K. A.; Schramm, V. L. J. Am. Chem. Soc. 1994, 116, 6531–

6536.

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A R T I C L E S

Characterization of Steady-State [Carbonyl-¹⁴C]nico-

tinamide Base-Exchange Reactions. Reactions containing 400

µM of NAD⁺, 250 µM of p53A with varying concentrations of

[carbonyl-¹⁴C]nicotinamide were initiated by addition of Af2Sir2

(ﬁnal concentration, 4.1 µM). Reactions were incubated for 90

min at 37 °C and quenched with 10% TFA to pH 2. After

centrifugation, clariﬁed reaction mixtures were injected on

HPLC (0.1% TFA eluent) to separate nicotinamide and NAD⁺.

Collected fractions containing nicotinamide and NAD⁺were

assayed for radioactivity by scintillation counting. Rates were

expressed as cpm/s incorporated into NAD⁺, and converted to

a turnover rate (1/s) after adjustment for enzyme concentration

and speciﬁc radioactivity. Rates were plotted versus nicotina-

mide concentration and ﬁt to the Michaelis-Menten equation

with Kaleidagraph.

combined in mixtures of [1′_N-H³]-, [8_A-¹⁴C]-NAD⁺’s and p53A.

[1′_N-H³] KIEs were measured as described above. Forward and

reverse commitment factors govern observed KIEs and are

predicted by Northrop’s eq 2:

KIE_obs) (KIE_int+ C_f+ C_r· KIE_eq)/(1 + C_f+ C_r)

(2)

where KIE_obsis the observed KIE, KIE_intis the intrinsic KIE

for the chemical step in question, C_fand C_rare the forward

and reverse commitment factors, respectively, and KIE_eqis the

equilibrium KIE separating reactants from the products of the

reaction step in question. With forward commitment negligible,

eq 2 is reduced to eq 3:

KIE_obs) (KIE_int+ C_r· KIE_eq)/(1 + C_r)

(3)

KIE Measurement. A mixture of ³H- and ¹⁴C-labeled NAD⁺

(total 300,000 cpm each) and peptide was divided into three

portions. The ﬁrst portion (420,000 cpm total) was diluted into

100 mM phosphate buffer (pH 7.5) containing unlabeled NAD⁺

to a ﬁnal concentration of 400 µM NAD⁺with 250 µM p53A

peptide in a volume of 80 µL. Reaction was initiated by addition

of Af2Sir2 (ﬁnal concentration ) 4.1 µM). Reaction was stopped

when 20% complete (45-60 min), and terminated by addition

of 10% TFA to pH 2.0. To convert AADPR to ADP-ribose

(ADPR), the quenched reaction was neutralized to pH 7.5 by

addition of 3 M NaOH and 1 M phosphate buffer (pH ) 9).

One unit of porcine liver esterase was added, followed by

incubation at 37 °C for 2 h. Reaction was quenched by addition

of 8 µL of 10% TFA. The second portion (90000 cpm) was

incubated at 37 °C for 1 h in the absence of Af2Sir2 as the

background control to detect impurity and/or nonspeciﬁc

hydrolysis of NAD⁺and was treated similarly with esterase.

The third portion (90000 cpm) was used for a 100% hydrolysis

reaction using CD38 (prepared as described⁵⁸) to hydrolyze

NAD⁺to ADPR. ADPR and remaining NAD⁺were separated

on a C18 column, and fractions containing ADPR were collected

and measured to precise 2 mL volumes with 15 mL of scintillant,

and the ³H/¹⁴C ratios were determined by scintillation counting.

The “intrinsic” or experimental KIEs without correction for

commitments were determined by eq 1:

In the case of Af2Sir2-catalyzed reactions, eq 4 was derived

from eq 3 to ﬁt the [1′_N-H³] KIE data at different nicotinamide

(NAM) concentrations. A full derivation based on Scheme 3 is

in the Supporting Information.

KIE_obs) (KIE_int+ (KIE_eq· C_r· [NAM])/(K_d+ [NAM]))/

(1 + (C_r· [NAM])/(K_d+ [NAM])) (4)

K_dis deﬁned according to Scheme 3 as the binding constant

of NAM to the imidate complex. The observed KIEs are

corrected for the reverse commitment according to eq 5, and

intrinsic KIEs are listed in Table 1.

KIE_int) KIE_obs(1 + C_r) - KIE_eq· C_r

(5)

Independently, using the relation C_r) k₄/k₅, where k₄is the

reverse rate of nicotinamide return to NAD⁺from the imidate

complex (Scheme 3), k₅is the forward rate of reaction from

the fully nicotinamide complexed imidate (Scheme 3), the value

of C_rwas computed using Michaelis parameters for Af2Sir2

using the peptide substrate using the assumption that K_d≈ K_m

(nicotinamide) for base exchange and with the assumption that

k₅≈ k_deacetyland k₄≈ k_B.Eas described in the text.

Computational Modeling. The reactant-state model of NAD⁺

was derived from the crystal structure of the lithium salt of

NAD⁺, truncated to nicotinamide riboside (NR) with all

hydrogens included.⁶⁰The geometry of the NR portion was

optimized in Vacuo using hybrid density functional methods with

B1LYP/6-31G** level of theory implemented in Gaussian 03,⁶¹

freezing two dihedral angles to maintain the ribosyl 3′-endo

conformation. The transition state was modeled as an oxacar-

benium ionlike intermediate, with nicotinamide-leaving group

and a N-methylacetamide nucleophile placed at different

distances to the anomeric carbon. The starting structure for the

ribose portion of the transition state was derived from the crystal

structure of ribonolactone as described.^60,44,48The carbonyl

oxygen was replaced with hydrogen, the orientation of the 5′-

hydroxy group was modiﬁed according to the crystal structure

of Af2Sir2 bound NAD⁺, and all atoms were minimized at the

B1LYP/6-31G** level. The transition state model was adjusted

by systematically altering C1′-N1, C1′-nucleophile bond

lengths and C1′-C2′, C1′-H1′, C2′-H2′, and C1′-O4′ bond

angles and dihedral angles while allowing the rest of the

molecule to be optimized as described above. The same level

of theory and basis set were used for optimization of substrate

KIE(experimental) ) ln(1 - fR_f/R_o)/ln(1 - f)

(1)

where f is the fraction reaction progress and R_fand R_oare the

ratios of light to heavy isotope in product at partial and 100%

completion, respectively, corrected for background. The intrinsic

KIE was corrected by the factor 1.001 for cases where

[2,8_A-³H]-NAD⁺was used as the remote, except for the

[4′_N-¹⁸O] KIE. In this case the value was divided by 1.001. The

remote KIE for [2,8_A-³H]-NAD⁺was determined to be 1.001(4).

Corrections for isotopic purities for ¹⁸O and ¹⁵N isotope effects

were applied.

Forward Commitment to Catalysis. Forward commitment

factor was determined by the isotope trapping method previously

described.⁵⁹A full description of the experimental method is

found in the Supporting Information.

Reverse Commitment to Catalysis. Reverse commitment to

catalysis was determined by measuring the [1′_N-H³] KIE as a

function of nicotinamide concentration. Nicotinamide at con-

centrations 0, 100 µM, 500 µM, 1 mM, 2 mM, and 5 mM was

(58) Munshi, C.; Lee, H. C. Protein Expression Purif. 1997, 11, 104–110.

(59) Chen, X. Y.; Berti, P. J.; Schramm, V. L. J. Am. Chem. Soc. 2000,

122, 1609–1617.

(60) Rising, K. A.; Schramm, V. L. J. Am. Chem. Soc. 1997, 119, 27–37.

(61) Frisch, M. J.; et al. Gaussian 03, Revision E.01; Gaussian, Inc.:

Wallingford, CT, 2004.

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A R T I C L E S

Cen and Sauve

Scheme 3. Chemical Mechanism for Sirtuin-Catalyzed ADP-Ribosylation of Acetyllysine and Relevant Reaction Downstream Chemistry from

Imidate^a

^aLower scheme depicts kinetic mechanism, including relevant rate constants and deﬁnitions of forward and reverse commitments relevant to the V/K KIE

measurement for the ADP-ribosylation of acetyllysine. The base exchange reaction described in the text occurs via reaction of NAD⁺forward to form the

imidate complex (EI₁), dissociation of nicotinamide from the EI₁·NAM complex, reassociation of radiolabeled nicotinamide to the complex (as shown by

fast equilibrium), reformation of NAD⁺via step governed by k₄, and release of reformed NAD⁺back into solution. Commitment equations relevant to

observed KIE values are found in Materials and Methods, and derivations for this kinetic mechanism are found in Supporting Information.

Table 1. Experimental and Intrinsic KIEs for Af2Sir2-Catalyzed

Deacetylation

good match to the experimental KIEs. Pauling bond orders were

calculated with the equation:

n_i) exp[(r₁- r_i)/0.3]

where r₁is the bond length of a single bond, and the

corresponding bond distances in the optimized structures were

used for the term r_iin the equation.

Calculation of Molecular Electrostatic Potential Surface. The

CUBE subprogram of Gaussian 03⁶¹was used to calculate

label of

interest

remote

label

experimental

KIE^a,b

intrinsic

KIE^c

molecular electrostatic potential (MEP) surfaces. The formatted

checkpoint ﬁles used in the CUBE subprogram were generated

by geometry optimization at the B1LYP/6-31G** level. The

MEP surfaces were visualized using Molekel 5.4⁶³at a density

of 0.2 electron/Å³.

Calculation of Group Partial Charges. The atomic and group

charges for N-methylacetamide, nicotinamide and ribose for both

reactant state and transition state were obtained using hybrid

density functional methods with B1LYP/6-31G** level of theory

implemented in Gaussian 03.⁶¹These data sets were generated

using the Gaussian 03 outputs for Mulliken atomic charges in

the initial computational steps for KIE calculation.

KIE type

primary

R-secondary

ꢀ-secondary

γ-secondary

δ-secondary

R-secondary

remote

d

1_N-¹⁵N

1′_N-¹⁴C

1′_N-³H

2′_N-³H

4′_N-³H

5′_N-³H

4′_N-¹⁸O

2,8_A-³H

8_A-¹⁴C

1.024(2)

1.014(4)

1.289(1)

1.095(5)

0.997(2)

1.019(5)

0.985(5)

1.001 (4)

1.024(2)

1.014(4)

1.300(3)

1.099(5)

0.997(2)

1.020(5)

0.984(5)

1.001(2)

^aKIE determined from at least three experiments and from correction

for isotopic depletion. ^bThe number in the parentheses represents the

error in the last digit. ^cKIE determined from experimental KIE using eq

3 and corrected for commitment factors of C_r) 0.038 and KIE_eq

)

Thermochemical Calculations of Transition State Ener-

gies. Geometries of the transition states were optimized in Vacuo

using hybrid density functional methods with B1LYP/6-31G**

level of theory implemented in Gaussian 03.⁶¹The same level

of theory and basis set were used for the computation of bond

frequencies, from which the free energy change was determined

for each molecule calculated. We calculated energies for

methylacetamide and nicotinamide riboside (calculated sepa-

rately), methyltriﬂuoroacetamide and nicotinamide riboside

(calculated separately), and three complexes, methylacetamide-

ribosyl-nicotinamide transition state (exo, as described in text

1.000 except for ﬁrst entry (See Supporting Information for KIEs

calculated for KIE_eq) 1.100). Error to the intrinsic KIE value includes

variability in the value of C_r(0.036-0.042) due to variability in the

20% conversion to products ((2%). ^dKIE determined from

experimental KIE using eq 3 and corrected for commitment factors of C_r

) 0.038 and KIE_eq) 1.027 (see reference for the equilibrium isotope

effect of 1.027 for full dissociation of nicotinamide). Measurement of

[1′_N-³H] KIE in the presence of nicotinamidase, which minimizes

reverse commitment by degrading nicotinamide, gave an experimental

KIE of 1.307(5) (corrected for isotope depletion of but without

correction of reverse commitment) in agreement with the calculated

intrinsic in Table 1.

as well as for the computation of bond frequencies. KIEs were

calculated from the computed frequencies using ISOEFF98.⁶²

The applied geometric constraints were optimized iteratively

until the KIEs predicted by the transition state model were a

(62) Anisimov, V.; Paneth, P. J. Math. Chem. 1999, 26, 75–86.

(63) Varetto, U. MOLEKEL Version; Swiss National Supercomputing

Centre: Manno, Switzerland.

9

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Transition State of Sirtuins

A R T I C L E S

Table 2. Calculated KIEs for Symmetric Transition State Models^a

with 2.65 Å distance to leaving group and nucleophile) the

corresponding isostructural methyltriﬂuoroacetamide-ribosyl-

nicotinamide complex (exo with 2.65 Å distance to leaving

group and nucleophile), and methylacetamide-ribosyl-nicotina-

mide, the fully dissociated oxacarbenium ion (exo, as described

in text with 3.5 Å C1′ distance to leaving group and nucleo-

phile). Energy of complex is relative to the reactant state.

Gaussian 03⁶¹provides outputs in energy in hartrees which were

converted to kcal/mol by the relation: 1 hartree ) 627.5 kcal/

mol.

3. Results and Discussion

symmetric models

NAD⁺Synthesis and Assays for Determining Isotope

Effects. To measure isotope effects for sirtuin chemistry we

chose the competitive radiolabel method.^44-47,53,54Isotopically

labeled NAD⁺’s were synthesized according to established

procedures⁵⁷except for synthesis of the [2′-³H]-NAD⁺which

required development of new synthetic methodology described

in the Supporting Information. Syntheses of the compounds,

[1′_N-³H], [4′_N-³H], [5′_N-³H], [1′_N-¹⁴C], [2,8_A-³H], [8_A-¹⁴C],

[4′_N-¹⁸O-2,8_A-³H], and [1-¹⁵N-5′_N-¹⁴C]-NAD⁺are described in

Materials and Methods and Supporting Information. Each

labeled NAD⁺was puriﬁed to homogeneity by HPLC, and

associative f dissociative

intrinsic KIEs

C_N1′-N_N1 (Å) 2.300 2.500 2.650* 2.675 2.725 3.55

C_N1′-Nu (Å) 2.300 2.500 2.650* 2.675 2.725 3.48

1_N-¹⁵N

1′_N-¹⁴C

1′_N-³H

2′_N-³H

4′_N-³H

5′_N-³H

4′_N-¹⁸O

1.023 1.025 1.025* 1.025 1.025 1.027

1.081 1.057 1.013* 1.012 1.010 0.998

1.309 1.411 1.424* 1.429 1.441 1.514

1.018 1.077 1.099* 1.106 1.117 1.196

0.994 1.002 1.003* 1.003 1.004 1.023

1.035 1.034 1.035* 1.035 1.036 1.029

0.995 0.995 0.993* 0.993 0.993 0.992

1.024(2)

1.014(4)

1.300(3)

1.099(5)

0.997(2)

1.020(5)

0.984(5)

^aIntrinsic KIEs are as reported in Table 1. Calculated KIEs are

determined as described in Materials and Methods. The numbers in the

ﬁrst two rows are bond distances in Å used in the calculation. The ring

geometry is 3′-exo, as explained in the text. Asterisked values represent

best ﬁt to experimental KIEs, shown in far right column.

checked for isotopic purity by MALDI-MS in cases where ¹⁵

and ¹⁸O labels were incorporated.

N

KIEs were measured by competing two distinctly isotopically

3

labeled substrate NAD⁺’s (one labeled ¹⁴C, the other, H) in a

is elevated, 1.139(5),⁵⁵as expected for an associative, nonox-

acarbenium ion transition state.

sirtuin reaction. Reactions were permitted to reach 20% product

formation, quenched, neutralized, and treated with esterase to

convert AADPR to ADPR. The experimental isotope ratio in

products was determined by HPLC collection of ADPR followed

by scintillation counting. A 100% reaction was generated by

converting NAD⁺to ADPR by CD38 (see Materials and

Methods for complete details). Experimental isotope effects

uncorrected for remote and commitment effects were computed

according to eq 1 in the Materials and Methods. (Corrections

for remote KIEs are described in the Materials and Methods,

and corrections for commitment factors are described further

in a later section.)

[1-¹⁵N] KIE. The primary leaving group KIE was measured

with [1-¹⁵N-5′_N-¹⁴C]-NAD⁺reacted against [2,8_A-³H]-NAD⁺,

wherein ¹⁵N is used to generate the primary isotope effect and

the ¹⁴C acts as the reporter. We demonstrated by mass

spectrometry that the ¹⁵N is incorporated to 99% (data not

shown). The intrinsic value of the KIE was 1.024(2) (Table 1).

The theoretical value for this KIE is 1.027 for a fully dissociated

transition state.⁴⁸A KIE value of 1.025 was computed for a

residual 0.0199 bond order remaining between nicotinamide and

the ribose ring of NAD⁺(Table 2).

[1′_N-³H] KIE. The R-secondary KIE is sensitive to rehybrid-

ization occurring at the anomeric carbon, speciﬁcally due to

changes in steric parameters affecting out-of-plane bending

modes at the transition state.^44-47,53,54For an associated

transition state of N-ribosyltransfer, where nucleophile and

leaving group are in a crowded pentacoordinate structure, steric

effects limit hydrogen bending and cause this KIE to be near

unity or even inverse.⁵⁵In cases where steric relief is apparent,

as in a dissociative transition state, the KIE is well above unity,

and for many oxacarbenium ionlike transition states for N-

ribosyltransfer reactions it has been measured in the range

1.16-1.36.^{44-47,53,54,59,64,65}For Af2Sir2 the [1′_N-³H] KIE is

1.300(3) on the very high end of reported values for this KIE

(Table 1). The R-secondary and the two primary KIEs indicate

an oxacarbenium-type transition state with signiﬁcant dissocia-

tion of the leaving group and nucleophile at the anomeric carbon.

The large value for the R-secondary KIE indicates that com-

mitments are not signiﬁcant and KIEs are nearly fully expressed.

The competitive method provides the V/K isotope effect, or

more plainly, the isotope effect for the ﬁrst irreversible step(s)

occurring on the enzyme. Conditions were identiﬁed wherein

the nicotinamide cleavage step was the one irreversible step

(discussed in the section on Commitments) using an acetyllysine

substrate (HLKSKKGQSTSRHK(K-Ac)LMFK) p53A reacted

with Archaeoglobus fulgidus Af2Sir2.

Kinetic Isotope Effects. The experimental KIEs corrected for

remote effects and for turnover are reported in Table 1. The

intrinsic KIEs on nicotinamide cleavage corrected for commit-

ments are also reported in Table 1. The analysis of commitments

follows a brief reporting and consideration of the intrinsic KIEs

for the reaction. Qualitative descriptions of the chemical and

structural inﬂuences that affect the values of these KIEs are

presented.

[1′_N-¹⁴C] KIE. The primary KIE at the anomeric carbon is

sensitive to the bonding occurring at this atom at the transition

state. This KIE was measured by competition of [1′_N-¹⁴C]-NAD⁺

against [2,8_A-³H]-NAD⁺. The remote tritium label on adenine

has a KIE of 1.001(4). The [1′_N-¹⁴C] KIE was determined to be

1.014(4) (Table 1). For dissociative N-ribosyltransfer mecha-

nisms the [1′_N-¹⁴C] KIE is close to unity and in the range 1.0

-1.03.^44-47,53,54For thymidine phosphorylase the [1′_N-¹⁴C] KIE

[2′_N-³H] KIE. The ꢀ-secondary KIE is sensitive to the

presence of a transition state orbital valency at the primary

(64) Singh, V.; Schramm, V. L. J. Am. Chem. Soc. 2006, 128, 14691–

14696.

(65) Lewandowicz, A.; Schramm, V. L. Biochemistry 2004, 43, 1458–1468.

9

J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010 12291

A R T I C L E S

Cen and Sauve

carbon^66,67In our case, the KIE is dependent upon the degree

of hyperconjugative overlap of the C-H bond at the ꢀ-position

with the orbital at the anomeric carbon at the transition state.

Hyperconjugation weakens the ꢀ-C-H bond and causes a

normal KIE. For solved oxacarbenium ionlike transition states

the ꢀ-secondary [³H] KIEs have ranged from 1.01-1.12.^48,59,67,68

Lower values are indicative of loss of the appropriate dihedral

angle for hyperconjugation, reaching the extreme when or-

thogonality deﬁnes the overlap.⁶⁹Low values can also result

from an associative mechanism where p-orbital development

at the primary carbon is less pronounced at the transition state,

thus preventing effective hyperconjugation.^55,67For Af2Sir2 the

[2′_N-³H] KIE was determined to be 1.099(5), at the high end of

this KIE for N-ribosyltransfer oxacarbenium ionlike transition

states,^48,67,69inconsistent with an associative transition state.⁵⁵

The value predicts that the ꢀ-hydrogen is in substantial overlap

with a largely vacant orbital at C1′ at the transition state.

[4′_N-¹⁸O] KIE. To obtain the value of the [4′_N-¹⁸O] KIE, we

competed [4′_N-¹⁸O-2,8_A-³H]-NAD⁺against [8_A-¹⁴C]-NAD⁺. The

KIE was 0.984(5) (Table 1), very close to the value obtained

for the solution hydrolysis of NAD⁺(0.988(7))⁴⁸and for

hydrolysis of NAD⁺catalyzed by diphtheria toxin of

(0.988(3)).⁴⁶The [4′_N-¹⁸O] KIE is inverse for oxacarbenium

ionlike transition states, because of double bond character at

the 4′_N-oxygen in a dissociated transition state.⁴⁸This effect is

closer to unity in associative mechanisms, although the value

for the [4′_N-¹⁸O] KIE for thymidine phosphorylase was not

reported.⁵⁵

[4′_N-³H] and [5′_N-³H] KIEs. Remote effects typically do not

deviate much from unity, and have somewhat limited value for

transition state determinations. On occasion, the 5′-position [³H]

KIE can be as high as 5%, although it appears the effect is

traced to binding isotope effects that propagate into the transition

state. In our case we report a modest [4′_N-³H] KIE of 0.997(2)

and a [5′_N-³H] KIE of 1.020(5).

Commitments. In this study, we determined the KIE under

conditions where the nicotinamide cleavage step is uniquely the

ﬁrst irreversible enzymatic step. The condition occurs when

binding steps are fully reversible (no forward commitment), and

if the nicotinamide cleavage step is not reversible (no reverse

commitment). The effects of forward and reverse commitments

on KIE values are described by eq 2. A forward commitment

means that binding is partially irreversible and a reverse

commitment means that the nicotinamide cleavage is partially

reversible. Commitments typically suppress the KIE and are

determined independently of the KIEs.

To measure forward commitment, the fraction of Michaelis

complexes that go forward irreversibly rather than dissociating

substrate (Scheme 3), we used isotope-trapping methods.⁵⁹

However, Af2Sir2 is so slow as a catalyst (<1 × 10^-2s^-1), we

did not expect an appreciable forward commitment. As shown

in Figure 1A, initial incubation with radiolabeled NAD⁺

followed by treatment with excess unlabeled NAD⁺does not

lead to trapped counts, and C_f) 0.000. Full details of this

experiment are provided in the Supporting Information and in

the Figure 1 legend.

Figure 1. (A) Measurement of forward commitment to catalysis for Af2Sir2

by the isotope-trapping method. Af2Sir2 was mixed with [8_A-¹⁴C]-NAD⁺.

After 10 s, the reaction was either terminated and analyzed for product

formation (circles) or diluted with chase solution containing a large excess

(10 mM) of unlabeled NAD⁺. Aliquots were removed at the indicated time

points and analyzed for product formation (squares). Controls used the same

amount of enzyme added directly to 10 mM NAD⁺solution containing

[8_A-¹⁴C]-NAD⁺, aliquots were removed and quenched at the indicated time

points (diamonds). The expected results for 50% commitment is shown as

a dotted line. (B) [1′-³H] KIE as a function of nicotinamide concentration

(circles). The curve was ﬁtted to the eq 4 in Materials and Methods. Values

ﬁt for the solid curve are shown in the ﬁgure. Independently, the Michaelis

parameters of base exchange and deacetylation were used to predict

commitment as discussed in the Materials and Methods, and the curve

predicting [1′-³H] KIE versus nicotinamide concentration is shown as the

dashed curve.

Reverse commitment is more pertinent because sirtuins are

known to reverse the imidate complex back to NAD⁺(Scheme

3).^39,40Af2Sir2 was chosen in light of our prior demonstration

of inefﬁcient nicotinamide reversal.⁴⁰To obtain the reverse

commitment, C_r, (eq 2) we considered the mechanism shown

in Scheme 3. The proposed mechanism assumes that nicotina-

mide is in fast binding equilibrium with the imidate complex

and that the forward chemistry terms k₅and k₅′ are nearly equal.

To determine that nicotinamide is in fast equilibrium compared

to chemistry we determined the steady-state base-exchange rate

as a function of solvent viscosity. The base-exchange rate is

measured with radioactive nicotinamide incubated in solution.

(66) Sunko, D. E.; Szele, I.; Hehre, W. J. J. Am. Chem. Soc. 1977, 99,

5000–5005.

(67) Cook, P. F. Enzyme Mechanisms from Isotope Effects; CRC Press:

Boca Raton, 1991.

(68) Singh, V.; Schramm, V. L. J. Am. Chem. Soc. 2007, 129, 2783–2795.

(69) Defrees, D. J.; Hehre, W. J.; Sunko, D. E. J. Am. Chem. Soc. 1979,

101, 2323–2327.

9

12292 J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010

Transition State of Sirtuins

A R T I C L E S

Base exchange is caused by release of nicotinamide from the

imidate complex followed by rebinding of radioactive nicoti-

namide. Imidate reversal forms radioactive NAD⁺followed by

radiolabeled NAD⁺release from the enzyme (Scheme 3, See

Materials and Methods for experimental). The steady-state base-

exchange rate is not sensitive to viscosity (Figure S1 Supporting

Information), consistent with rapid association and dissociation

steps for NAD⁺, acetyllysine substrate, and nicotinamide to the

enzyme compared to chemistry. To provide evidence that

complexes E·I₁·NAM and E·I₁react forward at similar rates,

the enzyme was incubated with elevated concentrations of

isonicotinamide, and base exchange and deacetylation were

assayed. At 20 mM, isonicotinamide inhibited base exchange

18% (Figure S2 Supporting Information) but did not inhibit

deacetylation (Figure S3 Supporting Information). Thus, ligand

occupancy of the nicotinamide site does not alter the forward

reaction parameter i.e. k₅≈ k₅′. Derivation of eq 4, which

describes commitments based in Scheme 3 is provided in the

Supporting Information.

To obtain the value for C_r) k₄/k₅, we determined the

[1′_N-³H] KIE as a function of nicotinamide concentrations. Fit

of points for the KIE as a function of nicotinamide concentration

was obtained using eq 4 (Figure 1B), and provided a value for

the intrinsic KIE, the value of C_r(k₄/k₅) and a value for K_d. The

determined intrinsic value for the [1′_N-³H] KIE of 1.300 is in

fair agreement to the observed experimental value of 1.289(1)

uncorrected for reverse commitment, and is in good agreement

with the KIE determined in the presence of nicotinamidase

1.307(5) (See Table 1). Nicotinamidase degrades nicotinamide

to nicotinic acid and prevents reaction reversal. The value of

C_rwas found to be 0.955 (at saturating nicotinamide concentra-

tions), and the value of K_d, the nicotinamide binding constant

to the imidate complex, was determined to be 0.95 mM.

Figure 2. Reaction coordinate diagram of Af2Sir2 catalyzed reactions. The

irreversible step is nicotinamide-glycosidic bond breaking and making (k₃

and k₄). (A) At saturating nicotinamide concentration, the maximum reverse

commitment was found to be 0.955. (B) The relevant reaction coordinate

during KIE measurements. Under these conditions the average nicotinamide

concentration is 40 µM, and the reverse commitment during experiment is

0.038.

Reverse commitment could also be estimated from the

Michaelis-Menten parameters for base exchange and deacety-

lation. For base exchange K_mwas determined to be 1.13 mM,

and the k_catwas determined to be 1.88 × 10^-3s^-1(See Materials

and Methods). For deacetylation, k_catwas measured to be 2.58

× 10^-3s^-1. Since deacetylation proceeds faster than base

exchange, we can infer that k₃> k₄and k₄< k₅(Scheme 3). Using

the assumptions k₄) k_cat(base exchange), k₅) k_cat(deacety-

lation) and K_d) K_m, C_r(k₄/k₅) is 0.729, in fair agreement with

the value obtained by curve ﬁt of the KIE values. The curve

for dependence of KIE versus nicotinamide from the Michaelis

parameters is shown in Figure 1B. For correction of KIEs, we

used C_r) 0.955, since it has no assumptions on k₄and k₅or K_d,

and was obtained by ﬁt of experimental data. The reaction

coordinate relevant for commitment with saturating nicotinamide

is shown in Figure 2A.

Transition State Determination by Gaussian and

ISOEFF98. The family of KIE data obtained is in good

correspondence with values previously determined for many

N-ribosyltransferases, and ADP-ribosyltransferases which sta-

bilize oxacarbenium ionlike transition states. To solve a specif-

ically deﬁned transition state for these KIEs, putative transition

states were computationally minimized at B1LYP/6-31G** level

of theory implemented in Gaussian 03, frequency sets computed,

and theoretical KIEs for each putative structure computed via

ISOEFF 98. Transition state structures were varied conforma-

tionally and in nucleophile and leaving group distances to match

calculated and experimentally determined KIEs. Bias was given

to matching the primary KIEs and the ꢀ-secondary KIEs, since

these KIEs were sensitive to nucleophile and leaving group

distances and the ribose ring conformation. We did not attempt

to match the R-secondary KIE since it did not vary across a

variety of models (see Tables 2 and 3) and it is difﬁcult to

reliably match computed and experimental R-secondary KIEs

for ribosyltransfer transition states.^59,64,68,70

To determine the effect of reverse commitment on KIE

measurements, consider that 400 µM NAD⁺was used with 20%

consumption. Under these conditions nicotinamide concentra-

tions average 40 µM, where the reverse commitment is 0.038

(from eq 4), considerably below the maximal value of 0.955.

The reaction coordinate relevant to conditions of KIE measure-

ment is shown in Figure 2B. Corrections to the intrinsic KIEs

using this commitment value are shown in Table 1. Resulting

corrections provide changes to the experimental values that are

very small and were used for computational determination of

the transition state structure of the nicotinamide cleavage step

catalyzed by the enzyme.

To calculate KIEs, a reactant reference structure and a putative

transition state structure were required. We chose the X-ray

crystallographically determined Li⁺NAD⁺structure as the

reactant,⁷¹as has been employed in previous studies of this kind.

(70) Scheuring, J.; Berti, P. J.; Schramm, V. L. Biochemistry 1998, 37,

2748–2758.

(71) Reddy, B. S.; Saenger, W.; Muhlegger, K.; Weimann, G. J. Am. Chem.

Soc. 1981, 103, 907–914.

9

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A R T I C L E S

Cen and Sauve

Table 3. Calculated KIEs for Asymmetric Transition State Models^a

middle).⁷³The ring conformation provides maximal hypercon-

jugative overlap of the 2′-position with a vacant orbital at C1′.

The 10% KIE for [2′_N-³H] KIE (Figure 3 right) required that

the dihedral angle be close to 0° (0.4°) to successfully model

the transition state.

Nucleophile-Associated Transition States. A series of calcula-

tions varying symmetrical distances (2.30 to 2.725 Å) of

nicotinamide N and acetyllysine carbonyl oxygen to C1′ were

performed (Table 2). Two computed KIEs were particularly

sensitive to these distances, the [1′_N-¹⁴C] KIE and the [2′_N-³H]

KIE. In a 2.3 Å symmetrical 3′-exo transition state, consistent

with a nucleophile-associated mechanism, the [1′_N-¹⁴C] KIE

isotope effect was calculated to be 1.081, and the [2′_N-³H] KIE

was calculated to be 1.018. These values are inconsistent with

the experimental values of 1.014(4) and 1.099(5), respectively,

for these KIEs.

asymmetric models

early T.S. f late T.S.

intrinsic KIEs

We modeled 3′-exo transition states that were asymmetrical,

with early or late character (Table 3). The 2.8 Å (LG), 2.35 Å

(NU) transition state model leads to a calculated [1′_N-¹⁴C] KIE

of 1.040 and a calculated [2′_N-³H] KIE of 1.107 outside errors

for the experimental KIEs. The 2.6 Å (LG), 2.35 Å (NU) model

proposed by QM/MM calculations⁵¹provided poor matches for

the [1′_N-¹⁴C] KIE and [2′_N-³H] KIE with calculated KIE values

of 1.054 and 1.081, respectively. We conclude that transition

states with less than 2.6 Å distance to the incoming acetyllysine

nucleophile (symmetric or asymmetric) are not supported by

our data.

C_N1′-N_N1 (Å) 2.500 2.650 2.675 2.600 2.800

C_N1′-Nu (Å)

2.800 2.700 2.650 2.350 2.350

1.026 1.025 1.025 1.027 1.028

1.047 1.013 1.012 1.054 1.040

1.453 1.430 1.427 1.397 1.418

1.093 1.103 1.105 1.081 1.107

1.001 1.003 1.003 0.999 1.002

1.037 1.035 1.035 1.037 1.037

0.998 0.993 0.993 0.995 0.995

1_N-¹⁵N

1.024(2)

1.014(4)

1.300(3)

1.099(5)

0.997(2)

1.020(5)

0.984(5)

1′_N-¹⁴C

1′_N-³H

2′_N-³H

4′_N-³H

5′_N-³H

4′_N-¹⁸O

^aIntrinsic KIEs are as reported in Table 1. Calculated KIEs are

determined as described in Materials and Methods. The numbers in the

ﬁrst two rows are bond distances (in Å) used in the calculation. The

ring geometry is 3′-exo, as explained in the text.

Dissociated Transition States. We considered an early transi-

tion state in which dissociation of the leaving group does not

occur prior to nucleophile association. This is reported in Table

3 as an asymmetric transition state, with bond distances of 2.5

Å (LG) and 2.8 Å (NU). The calculated [1′_N-¹⁴C] and [2′_N-³H]

KIEs were 1.047 and 1.093, respectively. Although the ꢀ-sec-

ondary KIE is within error, the poor match of [1′_N-¹⁴C] KIE

rules out an early transition state. The fully dissociated oxac-

arbenium ionlike structure, where bond orders were no greater

than 0.001 for both nucleophile and leaving group (3.55 Å (LG),

3.48 Å (NU)) (Table 2) provided calculated [1′-¹⁴C] and [2′-

³H] KIEs values of 0.998 and 1.196, respectively. The calculated

primary KIE is inverse, and the high calculated ꢀ-secondary is

well above the experimental value. The experimental data set

cannot be matched to a fully dissociated oxacarbenium ion.

Weak bonding participation of the nucleophile was required

at the transition state to match the primary and ꢀ-secondary

KIEs. We explored a range of symmetric and asymmetric

possibilities as shown in Table 2 and Table 3. Our best values

of bond distances were 2.65 Å (LG) and 2.65 Å (NU) at the

transition state (Table 2). The slightly more dissociated transition

state, 2.65 Å (LG), 2.70 Å (NU) in Table 3 also ﬁt the primary

and ꢀ-secondary KIEs, but less closely. These transition states

both match the intrinsic [1-¹⁵N], [1′_N-¹⁴C], and [2′_N-³H] KIEs

within error and have reasonable agreement with the [4′_N-¹⁸O]

and [4′_N-³H] KIEs. The geometric structures of solution reactant,

enzyme-bound Michaelis complex, and solved enzyme-bound

transition state (2.65 Å LG, 2.65 Å NU) are depicted in Figure

3.

Figure 3. Stick-and-ball models of reactant (left), Michaelis complex

(middle), and solved transition state (right; 2.65 Å leaving group distance,

2.65 Å nucleophile distance).

In turn, the transition state starting point was the geometry of

ribonolactone⁷²with subsequent replacement of the anomeric

carbonyl oxygen by hydrogen. The 5′-hydroxyl and 5′-carbon

were matched to the X-ray crystallographically determined

geometry of NAD⁺bound on Af2Sir2.⁷³To improve compu-

tational ease, we truncated the NAD⁺structure to nicotinamide

riboside (NR) as used previously for solution of an ADP-

ribosyltransfer transition state,⁶⁰and N-methylacetamide was

used as the reacting nucleophile. For a demonstration of the

reasonableness of the NR truncation, we present calculated KIEs

for two isogeometric transition state structures being different

only in having a 5′-OH or 5′-phosphate (see Supporting

Information, Table S5).

The reactant geometry of NAD⁺is 3′-endo (Figure 3 left),

but upon binding to a sirtuin, the NAD⁺adopts a 3′-exo ring

pucker as determined by X-ray crystallography (Figure 3,

It is instructive to examine the calculated and experimental

KIE values more in detail for the transition state structure that

we solved. The calculated values are shown in the asterisked

column of Table 2, and the experimental values are shown in

the far right column. The primary [1-¹⁵N] KIE determined

experimentally was found to be 1.024(2), whereas the optimal

model was calculated to be 1.025. The agreement is very good

(72) Kinoshita, Y.; Ruble, J. R.; Jeffrey, G. A. Carbohydr. Res. 1981, 92,

1–7.

(73) Avalos, J. L.; Bever, K. M.; Wolberger, C. Mol. Cell 2005, 17, 855–

868.

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12294 J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010

Transition State of Sirtuins

A R T I C L E S

Table 4. Bond Order and Bond Length for NAD⁺Reactant State and Optimal Transition State Structure and Fully Dissociated

Oxacarbenium for Reaction Catalyzed by Af2Sir2

reactant state

bond length (Å)

optimal transition state

oxacarbenium ion

bond length (Å)

bond

bond order

bond length (Å)

bond order

C_N1′-N_N1

C_N1′-O_N4′

C_N1′-C_N2′

C_N1′-H_N1′

C_N2′-H_N2′

C_N1′-Nu

1.533

1.381

1.540

1.092

1.099

0

0.824

1.101

0.954

0.993

0.970

0

2.65

0.0199

1.621

1.048

1.020

0.929

0.016

3.550

1.256

1.496

1.089

1.110

3.480

0.001

1.671

1.105

1.003

0.936

0.001

1.265

1.512

1.084

1.112

2.65

Bond lengths and bond orders are determined for the optimized structure of the symmetric transition state which best matches experimental intrinsic KIEs

and calculated KIE values as discussed in the text. Bond orders are Pauling bond orders and are calculated by methods in Materials and Methods.

and is consistent with a dissociated transition state structure,

with 0.0199 leaving group bond order to the anomeric carbon.

The primary [1′_N-¹⁴C] KIE experimentally determined to be

1.014(4) was calculated to be 1.013. The low KIE for the

primary carbon reﬂects low bond order to leaving group and

poor nucleophilic participation at the transition state, with bond

order of 0.016 between the anomeric carbon and the acetyl

carbonyl oxygen.

The low [1′_N-¹⁴C] KIE indicates compensatory bond stiffening

at this atom, in spite of the loss of the C1′-N1 bond, which

keeps the observed value near unity. In the optimal transition

state the calculated C1′-O4′ bond length is 1.265 Å (1.621 bond

order, Table 4) as compared with 1.381 Å (1.101 bond order)

in the reactant state. The bond order increases 0.520 at the

transition state. Stiffening of the C1′-O4′ bond is also

manifested in the inverse [4′_N-¹⁸O] KIE, calculated to be 0.993,

and experimentally determined to be 0.984(5).

The R and ꢀ secondary KIEs are interesting in comparison

to the values for the fully dissociated transition state structure.

In moving from a 2.65 to a 3.5 Å distance for both the leaving

and incoming group, the calculated [1′_N-³H] KIE increases from

1.424 to 1.514 (Table 2). The calculated value for the oxacar-

benium [1′_N-³H] KIE is near the theoretical limit for ADP ribosyl

Figure 4. Overlay of structures of Michaelis complex structure determined

from crystallography (red) and solved transition state (green; 2.65 Å leaving

group distance, 2.65 Å nucleophile distance). The structures are overlaid

with the pyridine N- position largely unchanged. The proposed displacement

of sugar anomeric carbon is determined to be 1.1 Å from the reactant C1′

position. The reactant acetyllysine oxygen was placed 3.6 Å from the

anomeric carbon of the reactant.

transfer. The calculated [2′_N-³H] KIE for the oxacarbenium ion

is 1.196 for a near perfect overlap of the C2′-H2′ bond with

the C1′ p orbital (0.4°) (Table 2), again near the theoretical limit

for ADP ribosyl transfer. The [2′_N-³H] KIE for the 2.65 Å

symmetrical structure was calculated 1.0998 in very good

agreement with the experimentally determined value of 1.099(5)

(Table 2).

Å from the transition state to the imidate complex with a C1′-O

bond distance computed to be 1.510 Å.⁵¹The total motion of

the sugar ring from reactants to products is 2.24 Å. Interestingly,

the 2′-OH migrates 1.8 Å from the NAD⁺-acetyllysine complex

to the thio-imidate on TmSir2.⁵²The ribose ring movement

reduces the bond angle of the nicotinamide N to the ribose ring

so that the N1 atom remains in optimal electronic overlap with

the developing p orbital at the transition state.

Michaelis Complex to Transition State Progression. An

overlay of the geometry of NAD⁺bound to Af2Sir2⁷³(truncated

to NR) with the determined transition state is shown in Figure

4. This overlay, which assumes nicotinamide N position is

virtually unchanged at the transition state is consistent with

“nucleophilic displacement by electrophilic migration”, as

suggested by recent structural studies.⁵²The reactant acetyl

oxygen at 3.6 Å to the reactant NAD⁺C1′, is in fair agreement

with the distance of of 3.2 Å reported in the NAD⁺-acetyllysine

termolecular structure of Thermatoga maritima Sir2.⁷⁴These

superpositions, with the assumption that the leaving group N

and acetyl O positions remain largely ﬁxed as proposed for

nucleophilic displacement by electrophilic migration,^52,75indicate

that the sugar anomeric carbon progresses 1.1 Å translationally

to form the transition state structure. The sugar translates 1.14

The sugar translation process causes the C2′-H2′ bond,

initially predicted to be -33.8° in dihedral angle to the N1-C1′

bond in the reactant complex, to narrow to 0.4° at the transition

state (Figure 5). This provides essentially maximal hypercon-

jugative overlap of the C2′-H2′ bond with the largely vacant

p orbital at the anomeric carbon in the transition state, and

explains the large ꢀ-secondary KIE. The initial 3′-exo reactant

ring geometry of NAD⁺in the reactant complex determined

crystallographically^73,74leads naturally to the solved transition

state geometry with ring ﬂattening and anomeric carbon

translation.

(74) Hoff, K. G.; Avalos, J. L.; Sens, K.; Wolberger, C. Structure 2006,

14, 1231–1240.

(75) Fedorov, A.; Shi, W.; Kicska, G.; Fedorov, E.; Tyler, P. C.; Furneaux,

R. H.; Hanson, J. C.; Gainsford, G. J.; Larese, J. Z.; Schramm, V. L.;

Almo, S. C. Biochemistry 2001, 40, 853–860.

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J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010 12295

A R T I C L E S

Cen and Sauve

Figure 5. (Left) 3′-Exo conformation of enzyme-bound reactant state, the N1-C1′-C2′-H2′ dihedral angle is -33.8°. (Right) 3′-Exo conformation of the

transition state, the p orbital C1′-C2′-H2′ dihedral angle is 0.4°.

Electrostatic Features of the Reactants and Transition

State. To visualize electronic changes that occur in the reactant

as it reaches the transition state we calculated the electrostatic

surfaces of reactant and transition state complexes (Figures 6

and 7, respectively) using Gaussian 03. In the reactant, H2, H6,

and C2 of the nicotinamide ring bear signiﬁcant positive charge

density prior to reaction. These charges are calculated to be

0.215, 0.218, and 0.189 respectively (See Supporting Informa-

tion for Table of calculated Mulliken charges for reactant and

transition state structures). These charges largely account for

the 0.541 charge on the pyridinium ring prior to reaction (Figure

8). The ribose ring also has positive charge density in the

reactant state (See Supporting Information), totaling 0.483

charge (Figure 8). The calculations of charge distribution are

in agreement with values determined for the pyridinium and

ribose ring in a prior computational study.⁵¹

At the transition state the nicotinamide is nearly neutral

(Figure 7). The net charge on the pyridine ring at the transition

state is reduced to 0.089 charge (Figure 8), a net charge loss of

0.452. The ribose ring gains charge, so that the total charge is

0.782 charge units at the transition state (Figure 8). The net

increase in charge on ribose in going from reactant to transition

state is 0.298. The greatest increase of charge occurs on the

O4′ atom (increase of 0.168), followed by the H2′, which

increases 0.085.

state. We were interested to understand how a weakly dissoci-

ated oxacarbenium-like transition state could be consistent with

both data sets. Therefore, we calculated the thermochemical

energies in Vacuo of the solved transition state structure, a fully

dissociated oxacarbenium ion (3.55 Å C1′-LG distance, 3.48

Å C1′-NU distance), and an isogeometric structure to the solved

transition state (2.65 Å symmetric) with N-methyltriﬂuoroac-

etamide (see Supporting Information Figure S4 for structures).

The solved transition state structure relative to reactants was

21.05 kcal/mol, but the fully dissociated oxacarbenium was

25.88 kcal/mol. Thus, calculations conﬁrm that the weakly

bonded transition state is intrinsically stabilized relative to the

fully dissociated oxacarbenium.

Mulliken charges for the fully dissociated oxacarbenium ion

(see Supporting Information for full charge tables) revealed that

the N-methylacetamide nucleophile bears a net charge of 0.094.

However, the charge on the fully dissociated ribosyl cation is

increased to 0.885 from 0.782 in the oxacarbenium ionlike

transition state. The weakly bonded transition state has increased

charge distribution to nicotinamide (charge on group: oxacar-

benium: 0.0207, TS: 0.0891) and N-methylacetamide (charge

on group: oxacarbenium: 0.0938, TS: 0.129) which could

provide some energetic stabilization.

Interestingly, the group charge on the acetyllysine increases

from fully neutral at the reactant state to 0.129 at the transition

state, even though only 0.016 bond order is calculated for the

transition state structure. Inspection of the atomic charges reveals

increased negative charge on the carbonyl oxygen (-0.518 in

the reactant to -0.599 at the transition state). The charge on

the nucleophile indicates a potent effect of the ribose cation on

charge distributions on the acetyllysine, even at weak bonding.

Increased partial positive charge density on the R-hydrogens,

amide N, amide H, and N-methyl hydrogens also results (see

Supporting Information). A similar charge transfer effect was

seen by Hu et al. in the calculation of the 2.6 Å (LG distance),

2.35 Å (NU distance) transition state model, where the bond

order was reported to be 0.15 at the transition state, the

acetyllysine charge was found to be approximately 0.15.⁵¹

Energies and Charge Distributions for Oxacarbenium and

Triﬂuoroacetyl Complexes. The ﬁnding that KIEs and calcula-

tion determine a weakly bonded oxacarbenium ionlike transition

state was very different from interpretations of Smith and

Denu,⁵⁰where very low rates of turnover with ﬂuorinated

acetyllysine derivatives led these authors to propose considerable

nucleophilic participation in the nicotinamide cleavage transition

The calculations with the triﬂuoro group were consistent with

experimental observations showing it reacts very slowly.⁵⁰The

calculated energy of the triﬂuoro structure was determined to

be 25.17 kcal/mol, destabilized 4.12 kcal/mol versus the isosteric

N-methylacetamide structure. The energy accounts for half

needed to explain the 600000-fold slower reaction of the

triﬂuoroacetyllysine versus the unsubstituted acetyllysine,⁵⁰

although our calculations explicitly ignore enzyme and solvent

effects. The slow rate could imply poor charge transfer from

the weaker nucleophile to the oxacarbenium ion. However,

surprisingly, the charge on the N-methyltriﬂuoroacetamide was

determined to be 0.121 charge units. Moreover the charges on

nicotinamide and sugar were 0.0918 and 0.787, very close to

the observed charges for the solved transition state with

unsubstituted N-methylacetamide. Thus, the charge distribution

per se does not deﬁne the energy of the complex. We suspect

that the interaction of the triﬂuoro nucleophile, which the

calculation indicates can still redistribute charge, is not stabiliz-

ing since the fully dissociated oxacarbenium ion and the

triﬂuoro-complex are only 0.71 kcal/mol different in energy.

9

12296 J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010

Transition State of Sirtuins

A R T I C L E S

Figure 7. Molecular electrostatic potential surfaces of transition state with

2.65 Å leaving group distance, 2.65 Å nucleophile distance (front top, side

middle, and back bottom). The transition state was determined in Vacuo

using the hybrid density functional methods implemented in Gaussian 03

using the B1LYP functional and 6-31G** basis set. The CUBE subprogram

of Gaussian 03 was used to generate molecular electrostatic potential (MEP)

surfaces, which are visualized using Molekel 5.4.

Figure 6. Molecular electrostatic potential surfaces of NAD⁺reactant state

(front top, side middle, and back bottom).

This result sets up the following scenario, proposed by Jencks,

for weak nucleophiles.⁷⁶He suggested that when a strong

nucleophile undergoes a substitution reaction at a reactive sugar

anomeric carbon, the reaction is more able to proceed via a

concerted mechanism.⁷⁶If the nucleophile is sufﬁciently

weakened, the reaction cannot be stabilized through preasso-

ciation (even in an “exploded” or highly dissociated transition

state), and thereby becomes stepwise. The results here can be

interpreted in this manner. For sirtuin chemistry, the acetyllysine

nucleophile preassociates in the substitution mechanism to the

extent of 0.016 bond order, with the leaving group still

associated to the extent of 0.019 bond order. The reaction

mechanism is a concerted one, although highly asynchronous,

since the leaving group is largely departed before the nucleophile

bond forms. The concerted mechanism of this type is highly

energetically favorable relative to the stepwise mechanism, as

shown by our energy calculations. However, when the sirtuin

nucleophile is switched to triﬂuoroacetyllysine, the calculations

suggest a much higher transition state barrier with little energetic

beneﬁt gained from nucleophile preassociation. In fact there is

little energy difference between the preassociated structure

(76) Banait, N. S.; Jencks, W. P. J. Am. Chem. Soc. 1991, 113, 7951–

7958.

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J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010 12297

A R T I C L E S

Cen and Sauve

deceleration observed for R-ﬂuoro-substitution to the 2-aceta-

mido group could be attributable to increased positive charge

on the pyranose ring at the transition state.⁷⁹That does not

appear to be the case here. A weaker nucleophile delivers similar

charge transfer to the sugar species as compared with a stronger

nucleophile in an isogeometric structure, but with appreciably

less overall stabilization.

4. Summary

The transition state structure of Af2Sir2 has been solved by

KIE and computational methods, and the solved transition state

structure shows that Af2Sir2 stabilizes an oxacarbenium ionlike

structure in the initial ADP-ribosylation of acetyllysine of

proteins with weak bond orders to nucleophile and leaving

group. The result is inconsistent with mechanistic proposals

invoking signiﬁcant nucleophilic participation (as measured by

bond order) at the transition state. The factors on the enzyme

that stabilize an oxacarbenium-like transition state are currently

poorly understood.⁷⁴Nevertheless, the weak acetyllysine and

nicotinamide association to the oxacarbenium structure provides

a signiﬁcant energetic stabilization relative to a fully dissociated

oxacarbenium ion. Computational studies provided insight into

the origins of signiﬁcant rate decelerations observed for ﬂuoro-

substituted acetyllysine nucleophiles. Characterization of other

transition states within this family of enzymes is expected to

determine how widely conserved the transition state structures

are and how much they can differ on sirtuins from different

sources. We suspect, given the high sequence conservation of

these enzymes, that sirtuins generally react highly dissociated

oxacarbenium ionlike transition state structures to ribosylate

acetyllysine residues to form nicotinamide and the ADP-

ribosylpeptidylimidate. It is anticipated that these transition state

structures can be used as blueprints for inhibitor design.

Figure 8. Computed group partial charges of acetyllysine, nicotinamide,

and ribose ring for reactant and transition state.

versus the dissociated oxacarbenium ion as predicted by the

Jencks argument. This result implies that the triﬂuoroacetyllysine

nucleophile could react via a stepwise substitution reaction.

A change to a stepwise mechanism opens the reaction

coordinate for alternative reaction outcomes. For example, there

is a highly conserved Phe residue that sits just over the ribose

oxygen, which could intercept the oxacarbenium to form a

cation-π complex⁵²rather than allowing ribosylation to the

triﬂuoroacetyl nucleophile. Since the nicotinamide is still the

best nucleophile in a competitive attack on the oxacarbenium

ion, the nicotinamide could rebound onto the oxacarbenium

before delivery to the triﬂuoroacetyllysine ever occurred. Such

“internal return” could provide additional rate slowing, which

in combination with a much higher barrier to nicotinamide bond

cleavage as calculated (in Vacuo 4.12 kcal/mol) could account

for the majority of the 600000-fold rate decrease observed for

triﬂuoro substitution. Importantly, almost none of the rate

difference is attributable to a mechanism requiring signiﬁcant

preassociation of the nucleophile, as has been suggested.⁵⁰There

is the additional possibility that a smaller part of the rate

difference arises from nonoptimal binding of triﬂuoroacetyll-

ysine to the active site.

Acknowledgment. This work was supported by NIH Grant R01

DK073466-5 to A.A.S.. We thank Piotr Paneth for providing

ISOEFF 98 and for a modiﬁcation to enable the software to work

with Gaussian 03 outputs.

Related work has been done in the reactions of ꢀ-N-

acetylglucosaminidases,⁷⁷which process substrates bearing

2-acetamide modiﬁcations. Fluorine substitution on the acetyl

group causes signiﬁcant rate deceleration, but not in each

case.^78,79Recent work has suggested that some of the rate

Supporting Information Available: Syntheses of materials,

calculations of KIEs and atomic charges of reactants and

transition states and derivation of reverse commitment equation

and additional experimental information are provided. Complete

references 35, 36, and 61 are available. This material is available

free of charge via the Internet at http://pubs.acs.org.

(77) Lairson, L. L.; Henrissat, B.; Davies, G. J.; Withers, S. G. Annu. ReV.

Biochem. 2008, 77, 521–555.

(78) Vocadlo, D. J.; Withers, S. G. Biochemistry 2005, 44, 12809–12818.

(79) Greig, I. A.; McCauley, M. S.; Williams, I. H.; Vocadlo, D. J. J. Am.

Chem. Soc. 2009, 131, 13415–13422.

JA910342D

9

12298 J. AM. CHEM. SOC. VOL. 132, NO. 35, 2010

Article Doi

Transition state of ADP-ribosylation of acetyllysine catalyzed by archaeoglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches

DOI: 10.1021/ja910342d

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Article abstract of DOI:10.1021/ja910342d

Full text of DOI:10.1021/ja910342d

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