A designed synthetic analogue of 4-OT is specific for a non-natural substrate

Article abstract of DOI:10.1021/ja050110b

The substrate specificity of 4-oxalocrotonate tautomerase (4-OT) is characterized by electrostatic interactions between positively charged arginine (Arg) side chains on the enzyme and the dianionic substrate, 4-oxalocrotonate. To generate specific hydrogen-bonding interactions with a monoanionic substrate analogue, we have introduced a urea functional group into the active site by replacing arginine side chains with isosteric citrulline (Cit) residues. This design was based on the complementarity between the urea functionality of citrulline and the uncharged amide function of the substrate, as opposed to the guanidinium-carboxylate electrostatic interaction between the wild-type enzyme and the natural substrate. Indeed, the synthetic (Arg39Cit)4-OT analogue catalyzed the tautomerization of the non-natural monoamide-monoacid substrate while it was a poor catalyst for the natural diacid substrate. The specificity of (Arg39Cit)4-OT for the monoamide-monoacid substrate relative to that of the diacid substrate was found to be 740-fold greater than that of the wild-type enzyme for tautomerization of the non-natural substrate as compared with the natural one. The role of electrostatic interactions in the tautomerization of the monoamide-monoacid substrate was probed in detail with several other Arg to Cit analogues of this enzyme. This study has demonstrated that chemical manipulation of the functional groups within the active site of an enzyme can modify its catalytic activity and substrate specificity in a predictable way, suggesting that the incorporation of noncoded amino acids into proteins has great promise for the development of new enzymatic mechanisms and new binding interactions.

Full text of DOI:10.1021/ja050110b

Published on Web 03/29/2005
A Designed Synthetic Analogue of 4-OT Is Specific for a
Non-Natural Substrate
Norman Metanis,†,‡,§ Ehud Keinan,*,†,‡,§ and Philip E. Dawson*,§,|
Contribution from the Department of Chemistry and Institute of Catalysis Science and
Technology, TechnionsIsrael Institute of Technology, Technion City, Haifa 32000, Israel, and
Department of Molecular Biology, Department of Cell Biology and Chemistry, and the Skaggs
Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
Received January 7, 2005; E-mail: dawson@scripps.edu (P.E.D.); keinan@tx.technion.ac.il (E.K.)
Abstract: The substrate specificity of 4-oxalocrotonate tautomerase (4-OT) is characterized by electrostatic
interactions between positively charged arginine (Arg) side chains on the enzyme and the dianionic substrate,
4-oxalocrotonate. To generate specific hydrogen-bonding interactions with a monoanionic substrate
analogue, we have introduced a urea functional group into the active site by replacing arginine side chains
with isosteric citrulline (Cit) residues. This design was based on the complementarity between the urea
functionality of citrulline and the uncharged amide function of the substrate, as opposed to the guanidinium-
carboxylate electrostatic interaction between the wild-type enzyme and the natural substrate. Indeed, the
synthetic (Arg39Cit)4-OT analogue catalyzed the tautomerization of the non-natural monoamide-monoacid
substrate while it was a poor catalyst for the natural diacid substrate. The specificity of (Arg39Cit)4-OT for
the monoamide-monoacid substrate relative to that of the diacid substrate was found to be 740-fold greater
than that of the wild-type enzyme for tautomerization of the non-natural substrate as compared with the
natural one. The role of electrostatic interactions in the tautomerization of the monoamide-monoacid
substrate was probed in detail with several other Arg to Cit analogues of this enzyme. This study has
demonstrated that chemical manipulation of the functional groups within the active site of an enzyme can
modify its catalytic activity and substrate specificity in a predictable way, suggesting that the incorporation
of noncoded amino acids into proteins has great promise for the development of new enzymatic mechanisms
and new binding interactions.
Introduction
analysis of enzymes that are well-characterized and has led in
some cases to altered enzymes with properties different from
The generation of new catalytic activities through structural
perturbation of existing enzymes is a powerful approach for
enzyme design.1 The resulting enzymes have potential as
biotechnologically useful catalysts, and these studies have led
to a better understanding of the molecular basis of enzyme
catalysis. One approach for the generation of new enzyme
activities is through screening of enzyme libraries generated by
methods of directed evolution.2-4 Alternatively, the rational
design of site-directed mutations5 within the enzyme active site
has been used successfully for the perturbation and mechanistic
those of the original wild-type enzymes.6 In addition, site-
specific chemical modification methods have enabled the
introduction of a broad range of functional groups into enzymes,
typically through modification of uniquely reactive amino acids
or cysteine side chains.7
In principle, the ability to introduce noncoded amino acids
into the active site of enzymes would facilitate the rational
design of new catalytic activities. The strategy of using
noncoded amino acids enables a precise placement of functional
groups that are typically not found in proteins into the binding
and catalytic machinery. This maneuver can be achieved by
either chemical synthesis of the enzyme or by a biosynthetic
approach.8,9 Using these methods, several noncoded amino acids
have been used to probe enzyme mechanisms.10-15
† TechnionsIsrael Institute of Technology.
‡ Department of Molecular Biology, The Scripps Research Institute.
§ The Skaggs Institute of Chemical Biology, The Scripps Research
Institute.
|| Department of Cell Biology and Chemistry, The Scripps Research
Institute.
4-Oxalocrotonate tautomerase (4-OT) is a 62 amino acid
homohexameric enzyme that is amenable to total chemical
synthesis.16 This enzyme is a member of a growing superfamily
(1) Harris, J. L.; Craik, C. S. Curr. Opin. Chem. Biol. 1998, 2, 127-132.
(2) For recent reviews, see: (a) Taylor, S. V.; Kast, P.; Hilvert, D. Angew.
Chem. Int. Ed. Engl. 2001, 40, 3310-3335. (b) Kraut, D. A.; Carroll, K.
S.; Herschlag, D. Annu. ReV. Biochem. 2003, 72, 517-571. (c) Piran, R.;
Keinan, E. In Catalytic Antibodies; Keinan, E., Ed.; Wiley-VCH: New
York, 2005; Chapter 9, pp 243-283.
(3) Farinas, E. T.; Bulter, T.; Arnold, F. H. Curr. Opin. Biotechnol. 2001, 12,
545-551.
(4) Stemmer, W. P. C. Nature 1994, 370, 389-391.
(5) Zoller, M. J.; Smith, M. Methods Enzymol. 1987, 154, 329-350.
(6) (a) Oxender, D. L., Fox, C. F., Eds.; Protein Engineering; Alan R. Liss,
New York, 1987. (b) Pick, L.; Hurwitz, J. J. Biol. Chem. 1986, 261, 6684-
6693.
(7) For recent review: Tann, C. M.; Qi, D.; Distefano M. D. Curr. Opin. Chem.
Biol. 2001, 5, 696-704.
9
5862
J. AM. CHEM. SOC. 2005, 127, 5862-5868
10.1021/ja050110b CCC: $30.25 © 2005 American Chemical Society

Specific Synthetic Analogue of 4-OT
A R T I C L E S
Scheme 1. A Proposed Mechanism of the Tautomerization of 4-Oxalocrotonate, 1a
a The three arginine residues Arg11′, Arg39′′, and Arg61′ (X ) NH2+) of the wild-type were replaced by citrulline (X ) O). The unprimed and singly
primed residues belong to different monomers of the same dimer, and the doubly primed residues belong to a third monomer from a neighbor dimer.
of enzymes that have been shown to catalyze isomerization,
decarboxylation, and dehalogenation reactions.17 These enzymes
utilize the secondary amine of the N-terminal proline as a
general acid or base as a common element in their catalytic
mechanisms. Mutation of this residue to glycine in 4-OT results
in a primary amine that can participate in both general acid/
base18,19 and nucleophilic catalysis.19,20 It has been shown that
4-OT catalyzes the tautomerization of a â,γ-unsaturated ketone,
2-oxo-4E-hexenedioate (4-oxalocrotonate, 1), to the R,â-un-
saturated isomer, 2-oxo-3E-hexenedioate (3), through a proposed
enolate intermediate, 2 (Scheme 1).21 Kinetic studies have shown
that this efficient enzyme transfers a proton from C-3 to C-5
using the N-terminal Pro residue as a general-base (pKa ∼
6.4).17,18,21-26 Interestingly, although all six active sites appear
to be structurally identical, suicide inhibitors have been shown
to initially alkylate only three subunits of the enzyme. This
suggests that, when interacting with substrates, all six active
sites may not be functionally equivalent.23
It has been proposed that the tautomerization reaction with 1
involves two steps, as illustrated in Scheme 1. Deprotonation
(step a) is followed by protonation (step b) with the two
corresponding transition states, TS1 and TS2, having ap-
proximately equal energy barriers.27 Since the keto and enol
forms of 1 coexist in aqueous solution in rapid equilibrium, the
first step (a) involves abstraction of a proton from either carbon
C-3 or the oxygen at the C-2 position of the substrate. This
step is catalyzed by the N-terminal secondary amine (Pro1). In
the second step (b), Pro1 donates a proton to the substrate at
carbon C-5. Accordingly, the two main features of the catalytic
machinery of this enzyme are Pro1, which acts as a general
acid/base, and Arg39′′, which provides electrostatic stabilization
for the negatively charged oxygen at the C-2 position in both
transition states and the enolate intermediate.28 The active site
4-OT has been previously studied by site-directed mutagenesis
and has proven to be robust toward modification. For example,
the mutants Arg11Ala and Arg39Gln were characterized by
NMR and were found to display little perturbation to the active
site.24
(8) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997, 289,
266-298.
(9) For recent reviews, see: (a) Thorson, J. S.; Cornish, V. W.; Barrett, J. E.;
Cload, S. T.; Yano, T.; Schultz, P. G. Methods Mol Biol. 1998, 77, 43-
73. (b) Wang, L.; Schultz, P. G. Chem. Commun. 2002, 7, 1-11.
(10) Hacheng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. U.S.A
1999, 96, 10068-10073.
(11) Chang, M. C. Y.; Yee, C. S.; Nocera, D. G.; Stubbe, J. J. Am. Chem. Soc.
2004, 126, 16702-16703.
(12) Cisneros, G. A.; Wang, M.; Silinski, P.; Fitzgerald, M. C.; Yang, W.
Biochemistry 2004, 43, 6885-6892.
(13) Judice, J. K.; Gamble, T. R.; Murphy, E. C.; de Vos Abraham, M.; Schultz,
P. G. Science 1993, 261, 1578-1581.
(14) Kienho¨fer, A.; Kast, P.; Hilvert, D. J. Am. Chem. Soc. 2003, 125, 3206-
3207.
The importance of electrostatics in the 4-OT catalytic
mechanism was recently probed using noncoded amino acid side
chains.28 Arginine was substituted at three positions in the active
site by citrulline, an uncharged isostere of arginine (Scheme
1). Interestingly, substitution at positions 11 and 39 of 4-OT
(15) Baca, M.; Kent, S. B. H. Proc. Natl. Acad. Sci. U.S.A 1993, 90, 11638-
11642.
(16) (a) Fitzgerald, M. C.; Chernushevich, I.; Standing, K. G.; Kent, S. B. H.;
Whitman, C. P. J. Am. Chem. Soc. 1995, 117, 11075-11080. (b) Fitzgerald,
M. C.; Chernushevich, I.; Standing, K. G.; Whitman, C. P.; Kent, S. B. H.
Proc. Natl. Acad. Sci. U.S.A 1996, 93, 6851-6856.
(17) Whitman, C. P. Arch. Biochem. Biophys. 2002, 402, 1-13.
(18) Stivers, J. T.; Abeygunawardana, C.; Mildvan, A. S.; Hajipour, G.; Whitman,
C. P.; Chen, L. H. Biochemistry 1996, 35, 803-813.
(24) Harris, T. K.; Czerwinski, R. M.; Johnson, W. H., Jr.; Legler, P. M.;
Abeygunawardana, C.; Massiah, M. A.; Stivers, J. T.; Whitman, C. P.;
Mildvan, A. S. Biochemistry 1999, 38, 12343-12357.
(25) Lian, H.; Whitman, C. P. J. Am. Chem. Soc. 1993, 115, 7978-7984.
(26) Czerwinski, R. M.; Johnson, W. H., Jr.; Whitman, C. P.; Harris, T. K.;
Abeygunawardana, C.; Mildvan, A. S. Biochemistry 1997, 36, 14551-
14560.
(27) Cisneros, G. A.; Liu, H.; Zhang, Y.; Yang, W. J. Am. Chem. Soc. 2003,
125, 10384-10393.
(28) Metanis, N.; Brik, A.; Dawson, P. E.; Keinan, E. J. Am. Chem. Soc. 2004,
126, 12726-12727.
(19) Brik, A.; D’Souza, L. J.; Keinan, E.; Grynszpan, F.; Dawson, P. E.
ChemBioChem 2002, 3, 845-851.
(20) Brik, A.; Dawson, P. E.; Keinan, E. Bioorg. Med. Chem. 2002, 10, 3891-
3897.
(21) Whitman, C. P.; Arid, B. A.; Gillespie, W. R.; Stolowich, N. J. J. Am.
Chem. Soc. 1991, 113, 3154-3162, and references therein.
(22) Chen, L. H.; Kenyon, G. L.; Curtin, F.; Harayama, S.; Bembenek, M. E.;
Hajipour, G.; Whitman, C. P. J. Biol. Chem. 1992, 267, 17716-17721.
(23) Taylor, A. B.; Czerwinski, R. M.; Johnson, W. H., Jr.; Whitman, C. P.;
Hackert, M. L. Biochemistry 1998, 37, 14692-14700.
9
J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005 5863

A R T I C L E S
Metanis et al.
Scheme 2. 4-Oxalocrotonate (1) and the Analogous
Scheme 5. Tautomerization of 4-Oxalocrotonate (1)
Monoamide-Monoacid Substrate, 4-Oxalamidocrotonate (4)
Scheme 3. Split Resin Assembly of the 4-OT Analoguesa
Scheme 6. Tautomerization of 4-Oxalamidocrotonate (4)
compound 1s2.5, 4.5, and 10.5 (Scheme 5)sand of compound
4s4.2 and 7.2 (Scheme 6). The corresponding UV absorption
maxima of 1 at various pH values were 302 nm (pH 2.3), 294
nm (pH 7.4), and 350 nm (pH 12.4). The absorption maxima
of 4 were 300 nm (pH 2.3), 298 and 349 nm (pH 7.4), and 349
nm (pH 12.4).
a Citrulline (Cit) was incorporated at positions 11, 39, and 61, R )
Arginine, C¸ ) Citrulline.
Mechanistic Considerations. The titration analysis indicates
that the enol function of 4b has a much lower pKa (7.2) than
that of 1b (pKa ) 10.5). As a result, under the experimental
conditions (pH 7.38) used for the 4-OT catalysis, 4 is expected
to exist predominantly in its enolate form, 9 (Scheme 6).
Experimental and computational studies suggest that both
transition states of the reaction with the natural diacid substrate
are similar in energy.21,27 Since the pKa of the 4-oxalamido-
crotonate substrate is significantly reduced, the first step is
expected to be rapid for the enol (4b) and nonexistent for the
enolate (9); as a result, it is likely that the second step is rate-
determining with these substrates. Since the enolate corresponds
to intermediate 2 in the proposed tautomerization mechanism
of 1, the second step (b) should become rate-limiting in the
case of 4. We propose that in step b, the general acid Pro1H+
transfers a proton to C-5, thus producing the ketoamide product
10, in analogy to the mechanism proposed for 1. Consequently,
neither the general base property of Pro1 nor the anionic
stabilization effect of Arg39′′ would be required for efficient
catalysis in the case of 4 (Scheme 7).
A change of the rate-determining step in enzymatic catalysis
as a function of the substrate is not unprecedented. The case of
R-chymotrypsin-catalyzed hydrolysis of amides and esters
represents a classic example: with an amide substrate the rate-
determining step is acylation of the catalytic serine residue to
form the covalent acyl-enzyme intermediate, while with an
ester substrate, the deacylation step of the intermediate becomes
rate-determining.32 In addition, mutant enzymes have been
shown to have a rate-determining step different from that
observed for the corresponding wild-type enzymes.12,33,34
Kinetic Analysis. Table 1 summarizes the kinetic parameters
of the catalytic tautomerization of 4 using eight analogues of
4-OT. For clarity, Table 2 highlights the comparison of RRR,
RC¸ R, and RC¸ C¸ with substrates 1 and 4. In the case of 4, the
kinetic parameters were measured using standard assay condi-
Scheme 4. Synthesis of 4-Oxalamidocrotonate (4)a
a Key: (a) concd HCl, reflux, 8 h; (b) SOCl2 reflux, 14 h; (c) (i)
HMDS, CH2Cl2, rt, 10 h, (ii) 5% H2SO4; (d) (i) aq NaOH (2 M) 16 h, (ii)
HCl (6 M).
resulted in a significant decrease of catalytic proficiency, sug-
gesting that the positively charged guanidinium group of
arginine binds the negatively charged acid groups of 4-oxalo-
crotonate 1 through predominantly electrostatic interactions.
These mechanistic insights suggested that the arginine-citrulline
substitutions could be harnessed for the design of a modified
enzyme specific for a new substrate with different electrostatic
properties than 1. In particular, Arg39Cit could form a produc-
tive binding interaction with a monoamide-monoacid substrate,
4, by hydrogen-bond complementarity rather than through
electrostatic interactions (Scheme 2). Here we demonstrate that
citrulline analogues of 4-OT are up to 740-fold more specific
than the wild-type enzyme for tautomerization of the ketoamide
substrate 4 compared to the natural substrate 1.
Results and Discussion
Synthesis and Characterization of Enzymes and Sub-
strates. All 4-OT analogues as well as the wild-type enzyme
(wt4-OT, RRR) were synthesized as described previously
(Scheme 3).28 4-Oxalamidocrotonate (4), was synthesized from
diethyl 2-hydroxy-2,4-hexadien-1,6-dioate (5) (Scheme 4),29 by
converting it to 6-hydroxycarbonyl-2-pyron (6) and then to the
corresponding acyl chloride (7).30 Reaction of the latter with
hexamethyldisilizane yielded amide 8,31 which was converted
to 4 by treatment with base and then with acid. Titration of
substrates 1 and 4 with base established the pKa values of
(32) Zerner, B.; Bond, R. P. M.; Bender, M. L. J. Am. Chem. Soc. 1964, 86,
3674-3679.
(29) Lapworth, A. J. J. Chem. Soc. 1901, 79, 1265-1284.
(30) Wiley, R. H.; Hart, A. J. J. Am. Chem. Soc. 1954, 76, 1942-1944.
(31) Pellegata, R.; Italia, A.; Villa, M.; Palmisano, G.; Lesma, G. Synthesis 1985,
5, 517-519.
(33) Thoma¨, N. H.; Evans, P. R.; Leadlay, P. F Biochemistry 2000, 39, 9213-
9221.
(34) Wetmore, S. D.; Smith, D. M.; Radom, L. ChemBioChem. 2001, 12, 919-
922.
9
5864 J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005

Specific Synthetic Analogue of 4-OT
A R T I C L E S
Scheme 7. Proposed General-Acid Mechanism for the 4-OT-Catalyzed Tautomerization of 4 (arginine, X ) NH2+; citrulline, X ) O)
Table 1. Kinetic Parameters for Synthetic Wt4-OT, RRR, and
Citrulline Analogues with 4-Oxalamidocrotonate 4
the reaction with 1 requires the neutral state of Pro1 (Scheme
8). In the wild-type enzyme the pKa of Pro1 is ∼6.4,35 and
kcat/KM
presumably, Pro1 has been evolutionary optimized to be 90%
1
1
1
a
analogue
KM
(µM)
kcat (s-
)
(M-
‚
s-
)
kcat/kuncat
neutral at physiological pH 7.4. In contrast, for the catalysis
with substrate 4 at pH 7.4, only 10% of this enzyme is available
in the required protonated state. If the protein concentration is
corrected for protonation state, the kcat becomes 10-fold higher,
or equal to that of the diacid reaction. The expected rate profile
as a function of pH would show low activity below pH 6, where
the enzyme would be protonated but the substrate would be
predominantly in the enol form, and also above pH 8, where
the enzyme would be deprotonated and the substrate would be
an enolate. These extremes suggest that the optimized pH for
this enzyme-catalyzed reaction would be between 6 and 8. These
considerations suggest that an appropriate redesign of the active
site to increase the pKa of the catalytic Pro1 residue would result
in increased concentration of Pro1H+ at neutral pH. For
example, the pKa of Pro1 in the mutant Phe50Ala has already
been shown to be ∼7.3 as compared with 6.4 in the wild-type
enzyme, probably due to the increased dielectric constant of
the active site.36
RRRb
C¸ RR
RC¸ R
RRC¸
C¸ C¸ R
C¸ RC¸
RC¸ C¸
C¸ C¸ C¸
90 ( 25
19 ( 5
130 ( 26
36 ( 10
nd
49 ( 14
125 ( 18
nd
290 ( 35
4.6 ( 0.4
72 ( 8
27 ( 2
>0.5
4.1 ( 0.4
21 ( 2
>0.5
3.1 × 106
2.4 × 105
5.6 × 105
7.4 × 105
nd
2.7 × 104
6.8 × 103
2.5 × 103
nd
431.0
8.3 × 104
1.7 × 105
nd
384.0
2.0 × 103
nd
a Uncatalyzed rate for 4: 1.064 × 10-2 s-1
.
b Coding refers to positions
11, 39, and 61 of the protein: R ) Arg, C¸ ) citrulline.
Table 2. Kinetic Parameters for Synthetic Analogues RRR, RC¸ R,
and RC¸ C¸ with the Substrates 4-Oxalocrotaonate (1) and
4-Oxalamidocrotonate (4)
kcat/KM
1
1
1
a
reaction
KM
(
µM)
kcat (s-
)
(M-
‚
s-
)
kcat/kuncat
RRRb + 1
RRR + 4
RC¸ R + 1
RC¸ R + 4
RC¸ C¸ + 1
RC¸ C¸ + 4
62 ( 10
90 ( 25
160 ( 40
130 ( 26
nd
2500 ( 150
290 ( 35
1.5 ( 0.2
72 ( 8
> 0.5
21 ( 2
4.0 × 107
3.1 × 106
9.8 × 103
5.6 × 105
nd
2.8 × 106
2.7 × 104
1.8 × 103
6.8 × 103
nd
Arg39Cit Is Specific for the Tautomerization of the
Ketoamide Substrate 4. In the reaction of 4 under the catalysis
of the Arg39Cit analogue, RC¸ R, kcat decreased only 4-fold with
insignificant change in KM (1.4-fold increase) in comparison to
catalysis with the wild-type enzyme, RRR (Table 2). Consistent
with our previous results, Arg39′′ has little effect on the KM of
either 1 or 4. In addition, RC¸ R showed similar kcat (only 4-fold
decrease) to that of RRR, suggesting that electrostatic effects
play a minor role in the transition state stabilization with 4, as
compared to the reaction with 1. Although KM is not necessarily
equal to a KD value, treatment of KM as an apparent dissociation
constant has been used previously in the context of 4-OT.24 This
result supports our proposed mechanism for the tautomerization
of 4. As the positive charge of Arg39′′ does not play a critical
role in the catalysis reaction with 4, deletion of this positive
charge causes only minor changes in kcat. The RC¸ R analogue
shows 740-fold greater specificity for the catalysis with 4 versus
1 compared to the wild-type enzyme, RRR. This result is also
supported by the double mutant, RC¸ C¸ , which exhibits a similar
125 ( 18
1.7 × 105
2.0 × 103
a Uncatalyzed rate for 1, 8.7 × 10-4 s-1; for 4, 1.064 × 10-2 s-1
.
b Coding refers to positions 11, 39, and 61 of the protein: R ) Arg, C¸ )
citrulline. Data for reactions with substrate 1 are taken from ref 28.
tions for 4-OT (20 mM NaH2PO4, pH 7.38) following the
disappearance rate of enolate 9 (λmax ) 350 nm). The kinetic
parameters for substrate 1 were taken from our previous work.28
Wt4-OT Catalyzes the Tautomerization of Both Sub-
strates. As can be seen in Table 2, the wt4-OT, RRR, catalyzes
the tautomerization of 4 8.5-fold slower than that of 1, with
little change in substrate binding. The overall effect is a 13-
fold decrease in kcat/KM. This minor change in KM indicates that
charge complementarity does not play a significant role in the
binding of either the carboxylate-1 or carboxamide-1 of either
the ketoacid substrate or the ketoamide substrate, respectively,
and either Arg39′′ or Arg61′. This hypothesis is strongly
supported by our previous studies of the RC¸ R and RRC¸
analogues with 1, where removal of the positive charge from
either Arg39′′ or Arg61′ resulted in a small increase in the KM
values, 2.5- or 4.2-fold, respectively.28 The modification of the
substrate from a negatively charged ketoacid to a neutral
ketoamide is analogous to arginine to citrulline substitutions in
the enzyme.
k
cat to that of RC¸ R. Mutant RC¸ C¸ represents a specific catalyst
for the nonnatural substrate, 4, as it shows no detectable catalysis
with the natural substrate, 1.
Arg11 Controls Substrate Orientation. The (Arg11Cit)-
4-OT analogue, C¸ RR, is a poor catalyst for 4, with kcat being
An interesting consequence of the change in the rate-
determining step is that the reaction with 4 requires the
availability of the protonated state of Pro1 (Pro1H+), whereas
(35) Stivers, J. T.; Abeygunawardana, C.; Mildvan, A. S.; Hajipour, G.; Whitman,
C. P. Biochemistry 1996, 35, 814-823.
(36) Czerwinski, R. M.; Harris, T. K.; Massiah, M. A.; Mildvan, A. S.; Whitman,
C. P. Biochemistry 2001, 40, 1984-1995.
9
J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005 5865

A R T I C L E S
Metanis et al.
Scheme 8. Similar Binding between the wt-4OT and Either the Natural Diacid 1 or Non-Natural Monoamide-Monoacid Substrate 9
Scheme 9. Two Alternative Binding Modes of 9 within the Active Site of Analogue C¸ RR
64-fold smaller in comparison with wt4-OT (Table 1). Interest-
ingly, however, as reflected by the small KM value, C¸ RR binds
4 more tightly than wt4-OT binds either 1 or 4. This is a rather
unexpected observation, considering the major role played by
Arg11′ in binding of the natural substrate through charge
complementarity with carboxylate-6, as manifested by the
observation that substitution to citrulline increased KM by 13-
fold.28 In contrast, if the orientation of 4 is similar to the natural
orientation proposed for 1 (Scheme 9), then the KM of C¸ RR
would be expected to be similar to that of 1. We propose that
a 180° switch in the binding orientation of 4 within the active
site could explain the decrease in both kcat and KM observed
with C¸ RR. The inverted orientation (Scheme 9) would place
the negatively charged carboxylate-6 of 9, the enolate of 4, in
close proximity to the positive charges of Arg39′′ and Arg61′
and would form hydrogen-bond complementarities between
carboxamide-1 of 9 and the amide functionality of Cit11′.
Although this binding mode would result in better substrate
binding, it adversely affects catalysis. The catalytic general acid
in Pro1 is no longer positioned at the optimal distance and in
the optimal orientation to react with the γ-carbon of the enolate.
The proposed inverted orientation is also consistent with the
kinetic parameters of the double mutant C¸ RC¸ , which exhibits
both reduced KM and kcat (Table 1). The importance of elec-
trostatic interactions for substrate binding is highlighted by the
measurable loss of catalytic activity in the C¸ C¸ R and C¸ C¸ C¸
analogues in which both Arg11′ and Arg39′′ are substituted with
the neutral Cit residue. An inverted substrate orientation in the
active site has been reported for other enzyme systems.37
Arg61 Has a Minor Role in the Tautomerization of Both
Substrates. Catalysis of the tautomerization of 4 with the
Arg61Cit analogue, RRC¸ , exhibited a 2.6-fold decrease in KM
in comparison with the wild-type, RRR. This small decrease
in KM could result from a productive hydrogen-bonding interac-
tion between the carboxamide-1 of 4 and the urea functionality
of Cit61′. In contrast to the case with the diacid substrate, where
RRC¸ had little effect on kcat,28 RRC¸ exhibited a 11-fold decrease
in kcat with 4 (Table 1). This moderate decrease in kcat suggests
that substrate binding to the Cit residue may result in nonoptimal
orientation of the substrate with respect to the catalytic Pro1
residue.
Conclusions
This work has demonstrated that chemical manipulation of
the functional groups within the active site of an enzyme can
modify its catalytic activity and substrate specificity in a
predictable way. To generate specific hydrogen-bonding interac-
tions with a designed substrate analogue, we have introduced a
urea functional group into the active site of 4-oxalocrotonate
tautomerase by replacing the natural arginine side chain with
an isosteric citrulline residue. This design was based on the
complementarity between the urea functionality of citrulline and
the uncharged amide function of the substrate, as opposed to
the guanidinium-carboxylate electrostatic interaction between
the wild-type enzyme and the natural substrate. Indeed, the
synthetic (Arg39Cit)4-OT analogue catalyzes the tautomeriza-
tion of a designed monoamide-monoacid substrate while it is
a poor catalyst for the natural diacid substrate. This work sug-
gests that the incorporation of noncoded amino acids into pro-
teins has great promise for the development of new enzymatic
mechanisms and new binding interactions.
Experimental Section
General Methods. 1H NMR spectra were recorded on a Bruker AM
200 or Bruker AM 500 spectrometers, using CDCl3 as a solvent (unless
otherwise specified). 13C NMR spectra were recorded on a Bruker
AV300 UltraShield with robot. Mass spectra were measured on a
(37) (a) Schwarz, K.; Borngraber, S.; Anton M.; Kuhn, H. Biochemistry. 1998,
37, 15327-15335. (b) Hornung, E.; Walther, M.; Kuhn, H.; Feussner, I.
Proc Natl Acad Sci U.S.A. 1999, 96, 4192-4197.
9
5866 J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005

Specific Synthetic Analogue of 4-OT
A R T I C L E S
Finnigan MAT TSQ700 (CI with isobutane) and infrared spectra on a
Bruker FTIR Vector22. Buffers for kinetic measurements were prepared
using deionized water. NaH2PO4 and Na2HPO4 were purchased from
Fisher Biotech. All Boc-amino acids were obtained from Midwest
Biotech (Fishers, IN), with the following side chain protecting groups:
Arg(Tos), Asp(OcHxl), Glu(OcHxl), Thr(Bzl), Ser(Bzl), Lys(2ClZ),
His(Dnp) (OcHxl ) cyclohexyl, Bzl ) benzyl, 2ClZ ) 2-chloroben-
zyloxycarbonyl, Dnp ) 2,4-dinitophenyl). The solvents N,N-dimeth-
ylformamide (DMF), dichloromethane, acetonitrile (ACN) were of high
purity (HPLC-grade) and purchased from Fisher. Trifluoroacetic acid
(TFA) was obtained from Halocabon Products (River Edge, NJ).
Anhydrous HF was purchased from Matheson Gas (Cucamonga, CA).
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate (HBTU), N,N-diisopropylethylamine (DIEA), and Boc-Arg-
OCH2-Pam resin were obtained from Peninsula Laboratories (Belmont,
CA).
Analytical reversed-phase HPLC was performed on a Hewlett-
Packard HPLC 1050 with 214 nm UV detection using Vydac C-18
columns (5 µm, 0.46 × 15 cm). Preparative reversed-phase HPLC was
performed on Waters HPLC system using Vydac C-18 columns (10
µm, 2.5 × 25 cm). Linear gradients of acetonitrile in water with 0.1%
TFA were used for all systems to elute bound peptides. The flow rates
were 1 mL/min (analytical) and 30 mL/min (preparative). Buffer A is
MilliQ water containing 0.1% TFA; buffer B is acetonitrile with 10%
water and 0.01% TFA. Electrospray ionization MS was performed on
an API-III triple quadruple mass spectrometer (Sciex, Thornhill, ON,
Canada). Peptide masses were calculated from the experimental mass
to charge (m/z) ratios from all of the observed protonation states of a
peptide by using MacSpec software (Sciex). Theoretical masses of
peptides and proteins were calculated by using MacProMass software
(Beckman Research Institute, Duarte, CA).
All synthetic proteins were folded as described previously,16a by
dissolving them (∼0.3 mg) in 1 mL of assay buffer (20 mM sodium
phosphate, pH 7.38) followed by incubation at room temperature for
2.5 h and ultracentrifugation to remove solid impurities. Circular
dicroism (CD) experiments were carried out on AVIV-62DS spec-
trometer using a microcuvette (1 mm optical path, 300 µL) and the
spectra recorded in the range of 200-260 nm. The spectra of all folded
mutant proteins were measured at 25 °C in phosphate buffer (20 mM,
pH 7.35), with filtered and degassed phosphate buffer as the blank
solution. The results as a plot of mean molar ellipticity per residue
([θ], deg‚cm2 dmol-1) versus wavelength in 1-nm increments for the
folded proteins were nearly identical to the plot obtained for the wild-
type enzyme, indicating that the mutations did not cause any gross
conformational changes in the synthetic proteins. The hexameric folded
structure of the enzymes was confirmed by gel filtration (data not
shown). The protein concentration was determined using the method
of Waddell.38 Kinetic experiments based on UV measurements were
carried out on Shimadszu UV-1601 spectrophotometer using a cuvette
(1 cm optical path, 1 mL capacity).
Chemical Peptide Synthesis. The polypeptide chain of the 62 amino
acid monomeric units of wt4-OT and its mutants were synthesized
manually by solid-phase peptide synthesis (SPPS) methods and in situ
neutralization protocols for Boc chemistry as described previously,39
on a 0.4 mmol scale using 11-fold excess of Boc-protected amino acids
(except for the noncoded amino acids norleucine and citrulline, which
were used in 3-fold excess). Synthesis was initiated with Boc-Nγ-tosyl-
L-arginine-4-(oxymethylphenylacetoamidomethyl) resin (Boc-L-Arg-
(Tos)-Pam resin) with the following side chain protecting groups:
Arg(Tos), Asp(OcHxl), Glu(OcHxl), Thr(Bzl), Ser(Bzl), Lys(2ClZ),
His(Dnp). After completion of chain assembly, the His(Dnp) groups
were deprotected by treatment of the Boc-peptide-resin with a solution
of 20% 2-mercaptoethanol and 5% DIEA in DMF. The entire
polypeptide was deprotected and cleaved from the resin by treatment
of the dry peptide-resin with HF and 4% v/ v p-cresol for 1 h at 0 °C.
The crude peptide product was precipitated and washed with cold
anhydrous ether, dissolved in 6 M guanidinium chloride (Gn‚HCl) at
pH 2.0, and immediately purified by preparative reversed-phase HPLC.
In all proteins, Met45 was replaced with norleucine to prevent oxidation
of the enzyme during sample handling.16b
Diethyl 2-Hydroxy-2,4-hexadien-1,6-dioate, 5. This diester was
prepared using a slightly modified procedure of Lapworth.29,30 Thus,
potassium (8.2 g, 0.22 mol) was slowly added to tert-butyl alcohol (80
mL) under argon. Diethyl ether (50 mL) was added and the mixture
was stirred for 15 min at 0 °C. A solution of diethyl oxalate (27.1 mL,
0.2 mol) in 20 mL ether was added slowly at the same temperature,
and the mixture was stirred for an additional 15 min. A solution of
ethyl crotonate (24.9 mL, 0.2 mol) in 20 mL ether was added slowly
and the mixture was stirred for 30 min at the same temperature. The
mixture was kept overnight at 4 °C to allow precipitation of the
potassium salt of 5. The product was collected by filtration and then
dissolved in 750 mL of ice water. Aqueous acetic acid (50%, 35 mL)
was added and the resultant precipitate was collected by filtration and
washed with cold water to give diester 5 in the form of a yellow
crystalline powder (23.21 g, 55%). IR (Nujol): 1710 (m), 1635 (m),
1474 (m), 1444 (m), 1371 (m), 1425 (m br), 1024 (w), 877 (m), 774
1
(m), 725 (m) cm-1. H NMR (200 MHz, CDCl3): δ 7.61 (dd, J )
15.6, 11.6 Hz, 1H), 6.43 (br s, 1H), 6.26 (d, J ) 11.6 Hz, 1H), 5.96 (d,
J ) 15.6 Hz, 1H), 4.32 (q, J ) 6.2 Hz, 2H), 4.20 (q, J ) 7.1 Hz, 2H),
1.34 (t, J ) 5.7 Hz, 3H), 1.24 (t, J ) 7.3 Hz, 3H). 13C NMR (75.47
MHz, CDCl3): δ 167.0, 165.1, 144.4, 137.2, 123.5, 108.7, 63.3, 60.9,
14.7, 14.5 ppm. MS (CI with isobutane): m/e 215 [MH]+, 169.
2-Hydroxymuconate, 1b. Diester 5 (1 g, 4.7 mmol) was dissolved
in 20 mL of sodium hydroxide (2.0 N), kept overnight at room
temperature, and then acidified with hydrochloric acid (6.0 M, in
cold water) up to pH ∼2.0. The resultant precipitate, which was
collected by filtration, was found to be 2-hydroxymuconate, 1b (0.5 g,
70%). IR (Nujol): 3397 (m), 1674 (m), 1639 (m), 1617 (m), 1259
1
(m), 1162 (m), 1096 (m), 877 (m), 777 (m), 723 (m) cm-1. H NMR
(200 MHz, CD3OD): δ 7.62 (dd, J ) 15.6, 11.7 Hz, 1H), 6.14 (d, J )
11.7 Hz, 1H), 5.85 (d, J ) 15.5 Hz, 1H). 13C NMR (75.47 MHz,
CD3OD): 170.9, 167.0, 148.6, 140.0, 122.6, 109.4. MS m/e 161, 159
[MH]+, 141.
6-Hydroxycarbonyl-2-pyron, 6. Diester 5 (3.6 g, 16.8 mmol) was
dissolved in concentrated hydrochloric acid (100 mL) and the mixture
was refluxed for 8 h as described previously.30 The resultant 6 was
collected by filtration (1.74 g, 74%). IR (Nujol): 1732 (m), 1690 (m),
1626 (m), 1237 (m), 1202 (m), 1161 (m), 1125 (m), 886 (m), 737 (m),
723 (m) cm-1. 1H NMR (200 MHz, CD3OD): δ 7.47 (dd, J ) 9.2, 6.6
Hz, 1H), 7.04 (d, J ) 6.5 Hz, 1H), 6.42 (d, J ) 9.2 Hz, 1H). 13C NMR
(75.47 MHz, CD3OD): 163.0, 162.5, 151.5, 144.9, 121.8, 112.0. MS:
m/e 141 [MH]+.
6-Chlorocarbonyl-2-pyron, 7. Carboxylic acid 6 (1.08 g, 7.71
mmol) was dissolved in thionyl chloride (10 mL) and the mixture was
refluxed for 16 h.30 Excess thionyl chloride was removed under reduced
pressure and the resultant crude acyl chloride, 7 (1.1 g, 90%) was taken
1
to the next step without purification. H NMR (200 MHz, CDCl3): δ
7.47 (dd, J ) 9.4, 6.6 Hz, 1H), 7.28 (d, J ) 6.6 Hz, 1H), 6.64 (d, J )
9.4 Hz, 1H).
6-Aminocarbonyl-2-pyron, 8. Following the general procedure of
amide formation,31 acyl chloride 7 (1.1 g, 6.94 mmol) was dissolved
in dry methylene chloride (15 mL) and the mixture was added to an
ice-cold solution of hexamethyldisilizane (5 mL, 20 mmol) in dry
methylene chloride (50 mL). Then the reaction mixture was stirred at
room temperature for 24 h. Methanol (1.5 mL) was added and the
resultant organic phase was washed with 5% sulfuric acid (2 × 20
mL) and then with saturated ammonium sulfate (2 × 20 mL), dried
over magnesium sulfate, and filtered. Removal of solvents under
reduced pressure afforded amide 8 in the form of white powder (0.92
(38) Waddell, W. J. J. Lab. Clin. Med. 1956, 48, 311-314.
(39) Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H. Int. J.
Pept. Protein Res. 1992, 40, 180-193.
9
J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005 5867

A R T I C L E S
Metanis et al.
g, 95%). IR (Nujol): 3339 (m), 3302 (m), 3187 (m), 1763 (m), 1732
(m), 1703 (m), 1670 (m), 1631 (m), 1614 (m), 1086 (w), 864 (m), 850
(m), 815 (m), 618 (m) cm-1. 1H NMR (200 MHz, DMSO-d6): δ 8.12
(br s, 1H), 7.88 (br s, 1H), 7.66 (dd, J ) 9.3, 6.6 Hz, 1H), 7.01 (d, J
) 6.6 Hz, 1H), 6.54 (d, J ) 9.3 Hz, 1H). 13C NMR (75.47 MHz,
DMSO-d6): δ 158.3, 158.0, 151.1, 142.2, 116.6, 104.8. MS m/e 140
[MH]+.
Enzymatic Activity with 2-Hydroxymucon-1-amide, 4b. Each
enzyme was assayed spectrophotometrically at 25 °C by monitoring
the disappearance rate of 9 (ꢀmax ) 6.97 × 103 M-1 cm-1 at 350 nm)
immediately following the addition of 4b to an assay mixture containing
enzyme appropriately diluted in phosphate buffer (20 mM, pH 7.38).
Final enzyme concentration varied from 6.6 nM (for wt4-OT, RRR)
to ∼1 µM (for (Arg11Cit,Arg39Cit,Arg61Cit)-4-OT, C¸ C¸ C¸ ). The
substrate concentration varied from 20 to 200 µM, and the kinetic
parameters KM and kcat were calculated from nonlinear regression data
analysis.
2-Hydroxymucon-1-amide, 4b. Compound 8 (1.0 g, 7.19 mmol)
was dissolved in sodium hydroxide (2 N, 50 mL). The mixture was
stirred overnight and then acidified with cold hydrochloric acid (6 M).
The resultant white solid was filtered, washed with cold methanol, and
air-dried to give 4b (0.67 g, 60%). IR (Nujol): 3459 (m), 3356 (m),
3184 (m), 1732 (m), 1683 (m), 1643 (m), 1622 (m), 1569 (m), 1622
Acknowledgment. We thank the Israel-US Binational Science
Foundation, the German-Israeli Project Cooperation (DIP)
(E.K.), The Alfred P. Sloan Foundation (P.E.D.) and the Skaggs
Institute for Chemical Biology for financial support. We also
thank Dr. Yael Balazs from Chemistry department, Technion,
for NMR assistance. E.K. is Incumbent of the Benno Gitter &
Ilana Ben-Ami chair of Biotechnology, Technion.
(m), 1540 (m), 1264 (m), 1163 (m), 1107 (w), 873 (m), 722 (m) cm-1
.
1H NMR (200 MHz, DMSO-d6): δ 7.74 (br s, 1H), 7.47 (br s, 1H),
7.63 (dd, J ) 15.3, 11.9 Hz, 1H), 6.16 (d, J ) 11.9 Hz, 1H), 5.88 (d,
J ) 15.3 Hz, 1H). 13C NMR (75.47 MHz, DMSO-d6): δ 168.0 (s),
165.1 (s), 150.1 (s), 138.5 (d), 120.6 (d), 104.0 (d) ppm. MS: m/e 158
[MH]+, 140.
Enzymatic Activity with 2-Hydroxymuconate, 1b. The kinetic
parameters reported in Table 1 were taken from our previous study.28
JA050110B
9
5868 J. AM. CHEM. SOC. VOL. 127, NO. 16, 2005