Remote binding energy in antibody catalysis: Studies of a catalytically unoptimized specificity pocket

Article abstract of DOI:10.1021/ja983017e

Binding interactions remote from the hydrolytic reaction center have been probed with substrate and phosphonate transition state analogues to understand how these types of interactions are used to promote catalysis in the 17E8 system. We find that the hapten-generated recogniton pocket in 17E8 has properties that are analogous to those of specificity pockets in enzymes. We have also found that there are specific requirements to form catalytically productive interactions between the side chain and the recognition pocket including conformation, size, and geometry. An additional requirement includes favorable simultaneous interactions between the side chain and binding pocket along with favorable interactions with the oxyanion hole. The 17E8 side chain recognition pocket seems to be less catalytically efficient than analogous pockets in enzymatic systems. The apparent binding energy gained from the methylene-pocket interactions in the 17E8 system is significantly smaller than those observed in natural enzymes. Furthermore, 17E8 does not use specific interactions in the recognition pocket to significantly affect catalytic turnover (k_cat) which is thought to be a trait of an unoptimized catalyst. Analysis of the crystal structure of the 17E8·hapten complex has allowed for the identification of differences between the active sites of 17E8 and several proteases. The identified differences give insight to the sources of the inefficient use of binding energy.

Full text of DOI:10.1021/ja983017e

1434
J. Am. Chem. Soc. 1999, 121, 1434-1443
Remote Binding Energy in Antibody Catalysis: Studies of a
Catalytically Unoptimized Specificity Pocket
Herschel Wade and Thomas S. Scanlan*
Contribution from The Departments of Pharmaceutical Chemistry and Cellular and
Molecular Pharmacology, UniVersity of California, San Francisco, California 94143-0446
ReceiVed August 21, 1998
Abstract: Binding interactions remote from the hydrolytic reaction center have been probed with substrate
and phosphonate transition state analogues to understand how these types of interactions are used to promote
catalysis in the 17E8 system. We find that the hapten-generated recogniton pocket in 17E8 has properties that
are analogous to those of specificity pockets in enzymes. We have also found that there are specific requirements
to form catalytically productive interactions between the side chain and the recognition pocket including
conformation, size, and geometry. An additional requirement includes favorable simultaneous interactions
between the side chain and binding pocket along with favorable interactions with the oxyanion hole. The
17E8 side chain recognition pocket seems to be less catalytically efficient than analogous pockets in enzymatic
systems. The apparent binding energy gained from the methylene-pocket interactions in the 17E8 system is
significantly smaller than those observed in natural enzymes. Furthermore, 17E8 does not use specific interactions
in the recognition pocket to significantly affect catalytic turnover (kcat) which is thought to be a trait of an
unoptimized catalyst. Analysis of the crystal structure of the 17E8‚hapten complex has allowed for the
identification of differences between the active sites of 17E8 and several proteases. The identified differences
give insight to the sources of the inefficient use of binding energy.
Introduction
several binding interactions that are responsible for 17E8’s
catalytic properties, including a well-defined pocket for the
n-butyl side chain of the transtion state analogue, and presum-
ably the transition state of the hydrolytic reaction (Figure 1).9
Removal of these side chain interactions results in a loss of
catalysis, suggesting a role for the 17E8 recognition pocket that
is similar to the role of specificity pockets used by natural
enzymes, especially those that utilize peptides or amino acids
as substrates.4,6,10-12
To study this hapten-generated recognition pocket and the
catalytic use of remote binding interactions in the 17E8
mechanism, a series of R-(formylamino)phenyl esters, all of
which contain different side chains (Figure 2), were synthesized
and used in the 17E8-catalyzed reaction (Scheme 1). In addition,
a series of corresponding R-(formylamino)phosphonates were
synthesized to probe binding interactions that would occur upon
the formation of the hydrolytic transition state or a nearby
tetrahedral intermediate (Figure 3). The results from these
experiments provide a conceptual framework for how these
remote interactions are used in the 17E8 mechanism and how
important these interactions are to 17E8 catalysis.13
The use of binding energy to achieve rate accelerations is
one feature that distinguishes enzymes from simple catalysts.
This notion of translating binding energy into catalysis has been
exploited in the generation of enzyme mimics such as catalytic
antibodies. These immunoglobulins have been generated against
haptens that are designed to create binding sites that use binding
energy to promote catalysis.1-3 So far, most of the catalytic
antibody design efforts have focused on the protein environment
proximal to the portion of the substrate that undergoes bond
changes. Although there is no doubt that this part of the active
site is an essential feature of a catalyst, nature has shown that
interactions that occur away from the site of bond cleavage can
be used to promote catalysis.4-6
Previous studies suggest that binding interactions away from
the catalytic center are essential for catalysis by the antibody
17E8.7 This antibody was generated against a phosphonate
monoanion and catalyzes the efficient, enantioselective hydroly-
sis of the phenyl ester of n-formylnorleucine.8 The crystal
structure of the17E8‚hapten complex indicates that there are
* To whom correspondence should be addressed.
(1) Wade, H.; Scanlan, T. S. Annu. ReV. Biophys. Biomol. Struct. 1997,
26, 461-493.
Experimental Section
Synthesis. General Methods and Reagents. Reactions requiring
anhydrous conditions were carried out in flame-dried glassware under
(9) Zhou, G. W.; Guo, J.; Huang, W.; Fletterick, R. J.; Scanlan, T. S.
Science 1994, 265, 1059-1064.
(10) Fersht, A. R. Proc. R. Soc. London B 1974, 187, 397-407.
(11) Fersht, A. R.; Dingwall, C. Biochemistry 1979, 18, 1245-1249.
(12) Page, M. I. Angew. Chem., Int. Ed. Engl. 1977, 16, 449-459.
(13) For an example of the design and functional characterization of an
antibody binding site that was designed to exhibit broad specificity at the
R-substituent of the amino acid substrate see: Tanaka, F., Kinoshita, K.,
Tanimura, R., Fujii, I. J. Am. Chem. Soc. 1996, 118, 2332-2339. The
relaxation of substrate specificity at this position was accomplished by
substituting the R-substituent with the linker.
(2) Shokat, K. M.; Schultz, P. G. Annu. ReV. Immunol. 1990, 8, 335-
363.
(3) Benkovic, S. J. Annu. ReV. Biochem. 1992, 61, 29-54.
(4) Jencks, W. P. Catalysis in Chemistry and Enzymology; Dover
Publications: Mineola, 1969.
(5) Jencks, W. P. Cold Spring Harbor Symp. Quant. Biol. 1987, LII,
65-73.
(6) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H. Freeman
& Co.: New York, 1985; pp 147-149.
(7) Wade, H.; Scanlan, T. S. J. Am. Chem. Soc. 1996, 118, 6510-6511.
(8) Guo, J.; Huang, W.; Scanlan, T. S. J. Am. Chem. Soc. 1994, 116,
6062-6069.
10.1021/ja983017e CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/05/1999

Remote Binding Energy in Antibody Catalysis
J. Am. Chem. Soc., Vol. 121, No. 7, 1999 1435
Figure 3. Phosphonate phenyl esters. (Top) Phosphonate skeleton.
(Bottom) Side chains. Side chains correspond to (1p-6p, 11p) the
homologous series and (7p-10p) the branched series.
Scheme 1. Esterolytic Reaction Catalyzed by 17E8 [(Boxed)
Putative High-Energy Tetrahedral Intermediate] and
Phosphonate Anion Used To Generate 17E8
The resins were washed and prepared according to manufacturers’
recommendations.
Figure 1. (a) Active site residues promixal to the n-butyl side chain
of 5p. The hapten is colored black. The nomenclature for residues
consists of (from left to right) the one letter amino acid code, residue
number, and designation for the light (L) or heavy (H) chain of the
antibody. The figure was generated with the MIDAS program.75 (b)
Solvent accessible surface of the 17E8 hapten binding site (light gray)
complexed with 5p (black). The surface was generated with a 1.4 Å
probe using the MIDAS program.76 The coordinates of the complex
were taken from the PDB file 1eap.
Synthesis of n-Formylated Amino Acid Ester Substrates, 1-14.
Formylation of Substrates. The amino acids were formylated in the
manner as decribed by Sheehan and Yang.14 A general procedure for
the preparation of the formylated amino acids is as follows: Acetic
anhydride (33 mL, 325 mmol) was added dropwise to a solution of
d/l-norleucine (1.3 g, 10 mmol) dissolved in 100 mL of formic acid.
After the addition of acetic anhydride, the solution was stirred at room
temperature for 3 h. The mixture was then concentrated by rotary
evaporation to yield a white powder. This crude material was carried
on directly to the esterification step.
Preparation of Phenyl Esters. The phenyl esters were prepared
from the formylated amino acids as described by Castro et al.15
A
general procedure for the esterification of the formylated amino acids
is as follows: racemic n-formylnorleucine (1 g, 6 mmol), phenol (0.6
g, 6 mmol), triethylamine (1.2 g, 12 mmol), and HBTU (2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)
(2.3 g, 6 mmol) were dissolved in 50 mL of dichloromethane. The
mixture was stirred at room temperature for 3 h. Saturated aqueous
NaCl (50 mL) was added to the solution. The solution was then
extracted with ethyl acetate (3 × 30 mL). The organic phase was then
washed with 2 N HCl (3 × 30 mL), saturated NaHCO3 (3 × 30 mL),
and saturated NaCl (2 × 50 mL). The organic phase was then dried
with NaSO4 and the solvent removed by rotary evaporation to yield a
yellow oil, which was chromatographed. Elution of a gradient of (1:5
to 1:1) ethyl acetate-hexanes afforded 5 (1.2 g, 86% yield) as a
colorless oil.
Figure 2. Phenyl esters used to probe the catalytic transition state
binding interactions in the 17E8 mechanism. (Top) Substrate skeleton
where R represents the position at which the side chain (bottom) was
varied. Substrates with side chains correspond to (1-7) the homologous
series, (8-11) the branched series, and (12-14) the conformationally
restricted series.
n-Formylglycine Phenyl Ester (1). 1H NMR (300 MHz, CDCl3) δ
8.32 (s, 1H), 7.42 (t, J ) 7.5 Hz, 2H), 7.25 (t, J ) 7.8 Hz, 2H) 7.12
(d, J ) 7.8 Hz, 1H), 6.29 (br s, 1H), 4.39 (d, J ) 5.4 Hz, 2H); 13C
NMR δ 161.2, 129.6, 126.4, 121.2, 40.1; MS (EI) 180.1 (MH+), 94.0,
86.0, 65.0, 58.0; HRMS calcd for C9H10N1O3 180.0661, found 180.0662.
an atomosphere of argon. Anhydrous solvents were purchased from
Aldrich. Chromatography solvents were purchased from Fisher Corp.
and were used as received. Reagents were purchased from either Sigma
or Aldrich and used as received unless noted otherwise. 1H NMR and
13C NMR were recorded on a General Electric 300-MHz instrument.
The NMR samples were prepared in 5-mm tubes, and the chemical
shifts are reported in parts per million (δ) relative to the standards TMS
for samples in CDCl3 and TSP (3-(trimethylsilyl)-1-propanesulfonic
acid sodium salt) for samples in D2O. Flash chromatography was
performed with Merck silica gel 60 (230-400 mesh). Ion exchange
chromatography was performed with diethylaminoethyl (DEAE) Sepha-
dex A-25 (anion exchange) and Dowex 50WX2-100 (cation exchange).
1
n-Formylalanine Phenyl Ester (2). Yield 0.73 g (88%); H NMR
(300 MHz, CDCl3) δ 8.23 (s, 1H), 7.40 (t, J ) 7.8 Hz, 2H), 2.23 (t, J
) 7.5, 7.2 Hz, 1H), 7.10 (d, J ) 8.1 Hz, 2H), 6.35 (br s, 1H), 4.93 (p,
J ) 7.2 Hz, 1H), 1.62 (d, J ) 7.2 Hz, 3H); 13C NMR δ 160.5, 129.5,
126.3, 121.2, 47.0, 18.4; MS (EI) 194.1 (MH+), 165.1, 116.1, 100.0,
(14) Sheehan, J. C.; Yang, D.-D. H. J. Am. Chem. Soc. 1958, 80, 1154-
58.
(15) Castro, B.; Evin, G.; Claude, S.; Seyer, R. Synthesis 1977, 413.

1436 J. Am. Chem. Soc., Vol. 121, No. 7, 1999
Wade and Scanlan
94.0, 82.9, 72.0, 65.0, 55.0; HRMS calcd for C10H21N1O3 193.0739,
found 193.0748.
1.00 (m, 6H); 13C NMR δ 160.9, 160.7, 129.6, 126.3, 121.2, 55.1, 53.9,
38.2, 37.8, 15.6, 14.6, 11.6, 11.7; MS (EI) 236.1 (MH+), 142.1, 114.1,
94.0, 82.9, 69.1, 58.0; HRMS calcd for (MH+) 236.1287, found
236.1290.
n-Formyl-r-aminobutyric Phenyl Ester (3). Yield 0.64 g (80%);
1H NMR (300 MHz, CDCl3) δ 8.28 (s, 1H), 7.40 (t, J ) 7.8 Hz, 2H),
7.26 (t, J ) 7.5, 6.9 Hz, 1H), 7.10 (d, J ) 8.1 Hz, 2H), 6.29 (br s, 1H),
4.93 (q, J ) 6.9 Hz), 2.19-2.05 (m, 1H), 1.97 (h, J ) 7.2 Hz, 1H),
1.06 (t, J ) 7.5 Hz); 13C NMR δ 160.7, 129.6, 126.3, 121.2, 52.0,
25.7, 9.40; MS (EI) 207.1 (M+), 207.1, 179.1, 133.0, 114.1, 94.0, 86.1,
65.0, 58.1; HRMS calcd for C11H14N1O3 (MH+) 208.0974, found
208.0970.
n-Formyl-trans-crotylglycine Phenyl Ester (12). Yield 0.66 g
1
(89%). H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.39 (t, J ) 7.5,
7.8 Hz, 2H), 7.28 (t, J ) 6.9, 7.8 Hz, 1H), 7.08 (d, J ) 7.8 Hz, 2H),
6.17 (br s, 1H), 5.67 (d qt, J ) 15.0, 6.3 Hz, 1H), 5.42 (dt, J ) 15.0,
6.9 Hz, 1H), 4.96 (dt, J ) 7.8, 5.7 Hz, 1H), 2.71 (t, J ) 6.3 Hz, 2H),
1.72 (d, J ) 6.3 Hz, 3H); 13C NMR δ 160.4, 130.9, 126.3, 123.8, 121.2,
50.8, 35.3, 18.0; MS (EI) 233.1 (M+) 140.1, 112.1, 94.0, 82.9, 67.1;
HRMS calcd for C13H15N1O3 (M+) 233.1049, found 233.1052.
n-Formyl-cis-crotylglycine Phenyl Ester (13). The R-amino-cis-
4-hexenoic acid was synthesized by the method as described by
n-Formylnorvaline Phenyl Ester (4). Yield 0.69 g (90%); 1H NMR
(300 MHz, CDCl3) δ 8.26 (s, 1H), 7.39 (t, J ) 7.5 Hz, 2H), 7.26 (t, J
) 7.5, 7.2 Hz, 1H), 7.06 (d, J ) 8.1 Hz), 6.30 (br d, J ) 6.0 Hz, 1H),
4.95 (dt, J ) 7.5, 5.7 Hz, 1H), 2.09-1.97 (m, 1H), 1.87 (p, J ) 6.9-
7.5 Hz, 1H), 1.50 (sept, J ) 7.2, 7.5 Hz, 2H), 1.01 (t, 7.2 Hz, 3H); 13
C
O’Donnel et al. using cis-crotyl bromide.16 Yield 0.32 g (85%); H
1
NMR δ 160.7, 129.5, 126.3, 121.2, 50.8, 34.5, 18.5, 13.6; MS (EI)
222.1 (MH+), 128.1, 100.1, 94.0, 82.9, 65.0, 58.0; HRMS calcd for
C12H16N1O3 222.1130 (MH+), found 222.1133.
NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 7.41 (t, J ) 7.5, 7.8 Hz, 2H),
7.32 (t, J ) 7.5 Hz, 1H), 7.20 (d, J ) 7.8 Hz, 2H), 6.17 (br s, 1H),
5.70 (m, 1H), 5.43 (q, J ) 7.5 Hz, 1H), 4.97 (m, 1H), 2.80 (m, 1H),
2.70 (t, J ) 6.6 Hz, 1H), 1.70 (d, J ) 7.2 Hz, 1H); 13C NMR δ 160.6,
129.5, 129.1, 126.2, 122.8, 121.2, 50.6, 29.5, 13.0; MS (EI) 234.1
(MH+) 188.1, 154.1, 140.1, 112.1, 94.0, 84.1, 77.0, 67.1; HRMS calcd
for C13H15NO3 (M+) 234.1130, found 234.1129.
1-Amino-cis-4-hexenoic Acid. 1H NMR (300 MHz, CDCl3) δ 5.80
(m, 1H), 5.40 (m, 1H), 3.80 (p, J ) 6.3 Hz,1H), 2.68 (m, 1H), 2.58 (t,
J ) 6.6 Hz, 1H), 1.70 (d, J ) 6.9 Hz, 1H), 1.67 (d, J ) 6.9 Hz, 1H);
13C NMR d δ 174.0, 131.8, 130.0, 122.8, 121.9, 53.9, 54.0, 32.3, 27.5,
16.9, 11.9; MS (EI) 129.1 (M+), 129.1, 84.1, 74.1, 67.1, 55.1; HRMS
calcd for C6H11N1O2 (M+) 129.0790, found 129.0794.
n-Formylpropargylglycine Phenyl Ester (14). Yield 0.68 g (88%).
1H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 7.39 (t, J ) 7.5 Hz, 2H),
7.26 (t, J ) 7.2 Hz, 1H), 7.11 (d, J ) 7.8 Hz, 2H), 6.84 (br s, 1H),
5.08 (dt, J ) 7.8, 5.0 Hz, 1H), 2.96 (m, 2H), 2.80 (s, 1H); 13C NMR
δ 168.7, 160.7, 129.5, 126.4, 121.2, 72.2, 49.4, 38.5, 22.5; MS (EI)
235.2 (M+), 170.1, 142.1, 114.1, 94.0; HRMS calcd for C13H18N1O3
(MH+) 236.1287, found 236.1291.
Synthesis of n-Formylated Phenyl Phosphonates, 1p-10p. The
phosphonates were prepared by the route similar to that described by
Guo et al. The use of aldehydes with different side chains yielded the
series. A general procedure for the preparation of the phosphonates is
as follows: Triphenyl phosphite (10.0 g, 32 mmol), n-pentanal (4.0 g,
48.5 mmol), and benzyl carbamate (4.9 g, 32 mmol) were dissolved in
20 mL of glacial acetic acid. The solution was stirred at 50 °C for 2 h
and then stirred at room temperature for an additional 2 h. The solution
was then concentrated by rotary evaporation to yield a viscous, yellow
liquid. The liquid was dissolved in 120 mL of methanol and left a 4
°C overnight to yield white crystals (9.7 g, 65% yield).
The above condensation product (5 g, 11 mmol) was dissolved in a
solution of 30% HBr in acetic acid (5 mL), and stirred for 1.5 h at
room temperature. Water (100 mL) and ethyl ether (100 mL) were
added to the reaction, and the resulting mixture was stirred at room
temperature for 10 min. Sodium hydroxide (10 M) was added until the
aqueous phase reached pH 9. The organic phase was then washed twice
with water (50 mL). The addition of 4 M HCl (5 mL) resulted in the
precipitation of diphenyl R-amino alkyl phosphonate (3.5 g, 89% yield).
The amino hydrochloride salts were formylated as described by Chen
et al.17 Briefly, EDC (2.1 g, 11 mmol) was added to a solution of formic
acid (1.0 g, 22 mmol) in dichloromethane (30 mL) at 0 °C and stirred
for 15 min. A solution containing the hydrochloride salt (2.0 g, 6 mmol)
and N-methylmorpholine (1.1 g, 11 mmol) in dichloromethane (30 mL)
was added. The mixture was stirred at 0 °C for 8 h and then at 20 °C
for 10 h. The mixture was then washed with 1 N HCl (2 × 20 mL),
saturated NaHCO3 (2 × 20 mL), and saturated NaCl (1 × 20 mL).
After the organic phase was dried with NaSO4, the solvent was removed
by rotary evaporation to yield a yellow oil (1.5 g, 78% yield) which
was carried on directly to the next step.
n-Formylnorleucine Phenyl Ester (5). Yield 1.2 g (86%); 1H NMR
(300 MHz, CDCl3) δ 8.27 (s, 1H), 7.40 (t, J ) 7.8 Hz, 2H), 7.26 (t, J
) 7.2, 7.5 Hz, 1H), 7.09 (d, J ) 7.8 Hz, 2H), 6.21 (br d, J ) 6.0 Hz,
1H), 4.95 (dt, J ) 7.5, 5.7 Hz, 1H), 2.16-2.00 (m, 1H), 1.90 (m, 1H),
1.42 (m, 4H), 0.94 (t, J ) 6.9, 6.6 Hz, 3H); 13C NMR δ 160.7, 129.6,
126.3, 121.2, 51.0, 32.2, 27.2, 22.3, 13.8; MS (EI) 236.1 (MH+), 207.1,
142.1, 114.1, 94.0, 82.9, 69.1, 58.0; HRMS calcd for C13H18N1O3
236.1287 (MH+), found 236.1296.
n-Formyl-r-aminoheptanoic Acid Phenyl Ester (6). The R-ami-
nohepanoic acid was synthesized by the method as described by
1
O’Donnel et al using bromopentane.16 Yield 0.22 g (88%); H NMR
(300 MHz, CDCl3) δ 8.27 (s, 1H), 7.40 (t, J ) 7.5 Hz, 2H), 7.26 (t, J
) 7.5, 7.2 Hz, 1H), 7.09 (d, J ) 8.1, 2H), 6.19 (d, J ) 7.2 Hz, 1H),
4.95 (dt, J ) 7.5, 5.4 Hz, 1H), 2.05 (m, 1H), 1.88 (m, 1H), 1.49,1.30
(m, 8H), 0.89 (t, J ) 6.6 Hz, 3H); 13C NMR δ 160.5, 129.5, 126.3,
121.2, 51.0, 32.5, 31.3, 24.8, 22.4, 13.9; MS (EI) 250.1 (MH+), 156.1,
128.1, 94.0, 83.1, 58.0; HRMS calcd for C14H20N1O3 (MH+) 250.1443,
found 250.1440.
n-Formyl-r-aminohexanoic Acid Phenyl Ester (7). Yield 0.62 g
1
(88%); H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.42 (t, J ) 7.5
Hz, 2H), 7.26 (t, J ) 7.2 Hz, 1H), 7.10 (d, 7.8 Hz, 2H), 6.19 (br d, J
) 6.3 Hz, 1H), 4.9 (dt, J ) 6.9, 6.3 Hz, 1H), 2.05 (m, Hz, 1H), 1.87
(m, 1H),1.41, 1.31 (m, 8H), 0.90 (t, J ) 5.7 Hz, 3H); 13C NMR δ
MS(EI) 264.2 (MH+), 235.2, 170.1, 142.1, 114.1, 94.0, 77.0; HRMS
calcd for C15H22NO3 (MH+) 264.1600, found 264.1598
n-Formyl-R-aminoisobutyric Acid Phenyl Ester (8). Yield 0.47
g (60%); 1H NMR (300 MHz, CDCl3) δ 8.16 (s, 1H), 7.39 (t, J ) 7.5,
7.8, 2H), 7.24 (t, J ) 6.9, 7.2, 1H), 7.10 (d, J ) 7.8, 2H), 6.25 (br s,
1H), 1.75 (s, 6H); 13C NMR δ 160.5, 129.5, 126.0, 121.3, 56.2, 24.8;
MS (EI) 208.1 (MH+) 179.1, 147.0, 114.1, 94.0, 86.1, 65.0, 58.1;
HRMS calcd for C11H13NO3 (MH+) 208.0974, found 208.0983.
1
n-Formylvaline Phenyl Ester (9). Yield 0.58 g (75%); H NMR
(300 MHz, CDCl3) δ 8.38 (s, 1H), 7.46 (t, J ) 7.5, 7.8 Hz, 2H), 7.32
(t, J ) 6.6 Hz, 1H), 7.15 (d, 7.8 Hz, 2H), 6.21 (br d, J ) 6.0 Hz, 1H),
4.99 (dd, J ) 9.0, 4.5 Hz, 1H), 2.46 (h, J ) 6.9 Hz, 1H), 1.15, 1.12 (d,
J ) 6.9 Hz, d, J ) 7.2 Hz 6H); 13C NMR δ 170.3, 160.9, 150.3, 129.6,
126.3, 121.3, 55.7, 31.4, 19.0, 17.7; MS (EI) 222.2 (MH+), 128.1, 100.1,
94.0, 85.0, 72.1, 65.0; HRMS calcd for (MH+) 222.1130, found
222.1135.
n-Formylleucine Phenyl Ester (10). Yield 0.66 g (89%); 1H NMR
(300 MHz, CDCl3) δ 8.24 (s, 1H), 7.39 (t, J ) 7.5, 7.8 Hz, 2H), 7.25
(t, J ) 7.2 Hz, 1H), 7.10 (d, J ) 8.1 Hz, 2H), 6.30 (br d, 7.5 Hz, 1H),
4.99-4.92 (m, 1H), 1.90-1.67 (m, 3H), 1.02 (d, J ) 5.7, 6H); 13C
NMR δ 171.3, 160.8, 129.5, 126.2, 121.2, 49.5, 41.5, 24.9, 22.8, 21.9;
MS (EI) 236.1 (MH+), 207.1, 149.0, 142.1, 133.0, 114.1, 94.0 86.1,
77.0, 69.1; HRMS calcd for (MH+) 236.1290, found 236.1295.
n-Formylisoleucine Phenyl Ester (11). Yield 0.52 g (75%); 1H
NMR (300 MHz, CDCl3) δ 8.32, 8.30 (s, s 1H), 7.40 (t, J ) 7.5, 7.8
Hz, 2H), 7.26 (t, J ) 7.2, 7.5 Hz, 1H), 7.09 (d, J ) 8.1 Hz, 2H), 6.21,
6.15 (br d, br d, J ) 6.9 Hz, 1H), 5.07, 4.96 (dd, dd, J ) 9.0, 3.6 Hz,
J ) 8.7, 4.5 Hz, 1H), 2.17 (m, 1H), 1.57 (m, 1H), 1.34 (m, 1H), 1.06,
Diphenyl n-formyl alkyl phosphonate (1.5 g, 4.3 mmol) was
dissolved in a solution containing 4.5 mmol of NaOH, water (10 mL),
and dioxane (10 mL), and the resulting solution stirred at room
temperature overnight. The reaction mixture was then washed with ethyl
(16) O’Donnell, M. J.; Wojchiechowski, K. Synthesis 1984, 313-315.
(17) Chen, F. M. F.; Benoiton, N. L. Synthesis 1979, 709-710.

Remote Binding Energy in Antibody Catalysis
J. Am. Chem. Soc., Vol. 121, No. 7, 1999 1437
ether (10 mL), and the aqueous layer was dried by rotary evaporation.
The remaining residue was dissolved in 5 mL of water, brought to pH
8.5 with 2 N NaOH, and filtered through a 0.45 mm filter. The solution
was chromatographed on a DEAE column by eluting with a linear
gradient of 0-0.5 N (Et)3NH2CO3. The collected fractions were
concentrated by rotary evaporation and redissolved in water (3 mL).
The product was converted to the lithium salt by cation exchange
chromatography (Dowex-Li+ form). The collected fractions were
lyophilized, yielding 5p (Li+ salt) (0.79 g, 63% yield (29% overall))
as a white powder.
Phenyl [1-(1-N-Formylamino)alkyl]phosphonates. Methyl (1p).
Yield 0.38 g (25%); 1H NMR (300 MHz, D2O) δ 8.10 (s, 1H), 7.39 (t,
J ) 7.8 Hz, 2H), 7.21 (t, J ) 7.5 Hz, 1H), 7.16 (d, J ) 7.8 Hz, 2H),
3.62 (d, J ) 12.6 Hz, 2H); 13C NMR δ 166.1 (d, 5.6 Hz), 132.0, 126.7,
123.0, 37.0 (d, J ) 151.2 Hz), 17.8; LSIMS-MS(-) 214.1 m/z (MH-
- HCl).
3-Methylbutyl (9p). Yield 0.73 g (36%); 1H NMR (300 MHz, D2O)
δ 8.13 (s, 1H), 7.40 (t, J ) 7.8 Hz, 2H), 7.26 (d. J ) 7.5 Hz, 1H), 7.18
(d, J ) 7.8 Hz, 2H), 4.54 (dd, J ) 15.6, 10.8 Hz), 1.66 (m, 2H), 0.96
(d, J ) 5.4 Hz, 3H), 0.90 (d, J ) 5.4 Hz, 3H); 13C NMR δ 166.4 (d,
5.0 Hz), 132.4, 126.9, 123.4, 47.2 (d, J ) 152.8 Hz), 40.7, 27.0, 25.4,
23.0; LSIMS-MS(-) 270.0 m/z (MH- - HCl).
3,3-Dimethylbutyl (10p). Yield 0.37 g (22%); 1H NMR (300 MHz,
D2O) δ 8.12 (s, 1H), 7.39 (t, J ) 7.8 Hz, 2H), 7.20 (t, J ) 7.5 Hz,
1H), 7.15 (d, J ) 7.8 Hz, 2H), 4.35 (dd, J ) 16.8, 10.5 Hz, 1H), 1.85
(dd, J ) 14.7, 10.8 Hz, 1H), 1.58 (dd, J ) 13.8, 11.1 Hz, 1H), 0.91 (s,
9H); 13C NMR δ 166.0, 132.4, 126.8, 123.4, 46.5 (d, J ) 152.8 Hz),
45.0, 31.4; LSIMS-MS(-) 242.0 m/z (MH- - HCl).
1
Diphenyl Methyl Phosphonate. H NMR δ 7.34 (t, J ) 7.8 Hz,
4H), 7.20 (m, 6H), 1.80 (d, J ) 17.7 Hz, 3H) MS (EI) 248.1, 170.1,
155.0, 94.0, 77.0, 65.0; 13C NMR δ 150.2 (d J ) 8.1 Hz), 129.7, 125.1,
120.4, 11.4 (d, J ) 143.2 Hz); HRMS calcd for C13H13O3P1 248.0602,
found 248.0607.
N-Cbz-aminomethyl Phosphonic Acid. 1H NMR (300 MHz, D2O)
δ 7.32 (m, 5H), 5.08 (s, 2H), 3.45 (d, J ) 11.4 Hz, 2H); 13C NMR δ
137.7, 129.5, 129.1, 128.8, 67.9, 39.3 (d, J ) 151.5 Hz); MS (EI) 244.0
(M - H)+, 228.0, 197.1, 138.0, 108.1, 90.0, 79.1; HRMS calcd for
C9H12N1O5P1 (M+) 245.0453, found 245.0459.
Phenyl Methyl Phosphonate (11p). Yield 0.44 g (80%); 1H NMR
(300 MHz, D2O) δ 7.43 (t, J ) 7.5 Hz, 2H), 7.22 (t, J ) 7.8 Hz, 1H),
7.18 (d, J ) 7.5 Hz, 2H), 1.42 (d, J ) 16.5 Hz, 3H); 13C NMR δ 154.2
(d, J ) 6.9 Hz), 132.6, 127.1, 123.8, 14.7 (d, J ) 138 Hz); LSIMS-
MS(-) 171.0 (MH- - Li).
Antibody Preparation and Purification. 17E8 was isolated from
ascites fluid and purified by affinity chromatography (protein A) as
described by Guo et al.8
Diphenyl N-t-boc-aminomethyl Phosphonate. 1H NMR (300 MHz,
CHCD3) δ 7.18 (m, 5H), 7.06 (m, 10H), 4.97 (s, 2H), 3.82 (dd, J )
10.5, 6.0 Hz, 2H) 13CNMR d 129.8, 128.6, 128.3, 128.2, 125.5, 120.5,
67.5; MS (EI) 397.1 (M+), 304.1, 289.1, 256.1, 233.0, 215.0, 184.1,
170.1, 140.0, 107.0, 91.1, 77.0; HRMS calcd for C21H20N1O4P1 (M+)
397.1079, found 397.1065
Steady-State Kinetics of the Phenyl Esters. Michaelis-Menton
parameters for the substrates were determined by continuous measure-
ment at 270 nm (phenol release ꢀ ) 1400 M-1cm-1) using a Uvikon
930 (Kontron Instrument) UV-vis spectrophotometer. All assays were
performed with cuvette holders thermostated at 24.5 ( 0.5 °C with a
Lauda RM6 temperature control unit. Cells of 1 cm path length (0.5
mL) were used in each experiment. The buffer used in all kinetic
experiments was 50 mM borate-150 mM NaCl, pH 8.7. The antibody
concentrations used in the experiments ranged from 0.2 to 1.4 µM.
The concentration ranges of the substrates were as follows: (2, 3) 30
mM to 600 µM, (4, 5, 6, 7, 11, 12, 13, 14) 2.0 µM to 30 mM. All
substrates were soluble at these substrate concentrations. The reactions
were initiated by adding 20 µL of substrate stock in DMSO to a solution
of 13-25 µL of IgG (in PBS) and 455-467 µL of pH 8.7 borate buffer.
In the background reactions, the IgG was replaced with PBS. All
catalyzed assays were performed in triplicate. The background reactions
were performed in duplicate. The catalyzed rate was obtained by
subtracting the average of the background reaction rate from the rate
of the catalyzed reaction. The data (V vs [S]) from the experiments
were fit with the KaleidaGraph (Synergy Software) curve-fitting
program using the Michaelis-Menton equation.
Inhibition by the Phosphonates. The phosphonate inhibition
experiments were performed by obtaining the steady-state kinetic
constants, in an analogous manner as described above, in the presence
of fixed concentrations of the phosphonates. In these experiments, the
borate buffer contained the phosphonates. Hydrolysis of the phonspho-
nates in the buffer was not detected spectrophotometrically. The
background rate constants were identical within error to those measured
with the phosphonate-containing buffer. The phosphonate concentrations
ranged (1p, 10p) from 140 µM to 7 mM, (2p, 3p, 9p) from 30 to 300
µM (4p, 5p, 6p), from 1 to 30 µM, and (7p, 8p) from 120 to 600 µM.
The phosphonate solutions were either used upon preparation or
immediately stored at -20 °C. The norleucine substrate, 5, was used
in all of the inhibition experiments. The kinetic data were analyzed
with the use of Lineweaver-Burk plots. The inhibition constant was
obtained by plotting the slope (Km/Vmax(1 + [I]/Ki)) from the Lin-
eweaver-Burk plots against inhibitor concentration and obtaining the
intercept on the [I] axis (-Ki).18
1
Ethyl (2p). Yield 0.27 g (32%); H NMR (300 MHz, D2O) δ 8.05
(s, 1H), 7.38 (t, J ) 7.8 Hz, 2H), 7.20 (t, J ) 7.2 Hz, 1H), 7.15 (, J )
7.8 Hz, 2H), 4.47 (st, J ) 7.5 Hz, 1H), 1.55 (dd, J ) 15.9, 7.2 Hz,
3H); 13C NMR δ 165.7 (d, 5.5 Hz), 132.2, 126.7, 123.2, 43.9 (d, J )
153.2 Hz), 17.8; LSIMS-MS(-) 228.2 m/z (MH- - HCl).
Propyl (3p). Yield 0.59 g (44%); 1H NMR (300 MHz, D2O) δ 8.16
(s, 1H), 7.39 (t, J ) 7.5 Hz, 2H), 7.20 (t, J ) 7.2 Hz, 1H), 7.18 (d, J
) 8.1 Hz, 2H), 4.15 (ddd, J ) 14.7, 7.8, 7.2 Hz, 1H), 1.96 (m, 1H),
1.64 (m, 1H), 0.96 (t, J ) 7.2 Hz); 13C NMR δ 166.6 (d, 5.3 Hz),
132.3, 126.9, 123.4, 50.4 (d, J ) 152.0 Hz) 25.5, 13.0 LSIMS-MS(-)
242.0 (MH- - HCl).
1
Butyl (4p). Yield 0.24 g (30%); H NMR (300 MHz, D2O) δ 8.16
(s, 1H), 7.41 (t, J ) 7.5 Hz, 2H), 7.22 (t, J ) 7.5 Hz, 1H), 7.15 (d, J
) 7.5 Hz, 2H), 4.28 (ddd, J ) 14.7, 8.4, 7.2 Hz, 1H), 1.87 (m., 1H),
1.71 (m, 1H), 1.50 (m, 1H), 1.36 (m, 1H), 0.98 (t, J ) 7.2 Hz, 3H);
13C NMR δ 166.2 (d, 5.0 Hz), 132.2, 126.7, 123.2, 48.2 (d, J ) 152.3
Hz), 33.8, 21.4, 15.2; LSIMS-MS(-) 256.3 m/z (MH- - HCl).
Pentyl (5p). Yield 0.79 g (29%); 1H NMR (300 MHz, D2O) δ 8.02
(s, 1H), 7.37 (t, J ) 7.8 Hz, 2H), 7.18 (t, J ) 6.3 Hz, 1H), 7.15 (d, J
) 7.8 Hz, 2H), 4.22 (ddd, J ) 12.0, 8.4, 7.2 Hz, 1H), 1.91 (m, 1H),
1.65 (m, 1H), 1.31 (m, 4H), 0.98 (t, J ) 6.0 Hz, 3H); 13C NMR δ
166.2 (d, J ) 5.1 Hz), 132.1, 126.7, 123.1, 48.4 (d, J ) 152.2 Hz),
31.3, 30.1 (d, J ) 12.6 Hz), 23.9, 15.6; LSIMS-MS(-) 270.3 (MH-
Li).
-
Hexyl (6p). Yield 0.47 g (44%); 1H NMR (300 MHz, D2O) δ 8.14
(s, 1H), 7.40 (t, J ) 7.8 Hz, 2H), 7.21 (t, J ) 7.5 Hz, 1H), 7.17 (d, J
) 7.8 Hz, 2H), 4.35 (dd, J ) 16.8, 10.5 Hz, 1H), 1.91 (m, 1H), 1.66
(m, 1H), 1.44 (m, 1H), 1.10 (m, 5H), 0.87 (t, J ) 7.2, 3H); 13C NMR
δ 164.1 (d, 4.2 Hz), 132.1, 126.6, 123.1, 48.4 (d, J ) 152.8 Hz), 28.8,
27.5, 23.5, 20.2, 11.7; LSIMS-MS(-) 284.1 m/z (MH- - HCl).
1
2-Methylpropyl (7p). Yield 0.25 g (27%); H NMR (300 MHz,
D2O) δ 8.17 (s, 1H), 7.39 (t, J ) 7.5 Hz, 2H), 7.20 (t, J ) 7.5 Hz,
1H), 7.16 (d, J ) 8.4 Hz, 2H), 4.19 (dd, J ) 17.7, 4.0 Hz, 1H), 2.27
(m, 1H), 1.03 (d, J ) 6.9 Hz, 3H), 0.99 (d, J ) 6.6 Hz 3H); 13C NMR
δ 166.7 (d, J ) 6.1 Hz), 132.4, 126.9, 123.5, 53.7 (d, J ) 149.5 Hz),
31.2, 19.9; LSIMS-MS(-) 256.1 m/z (MH- - HCl).
2-Methylbutyl (8p). Yield 0.36 g (20%); 1H NMR (300 MHz, D2O)
δ 8.17,8.15 (s, 1H), 7.40 (t, J ) 7.5 Hz, 2H), 7.20 (t, J ) 7.8 Hz, 1H),
7.17 (d, J ) 7.5 Hz, 2H), 4.39,4.02 (dd, J ) 18.0, 4.8 Hz, 1H), 1.97
(m, 1H), 1.76 (m, 1H), 1.05, 1.01 (d, J ) 6.9 Hz), 0.91 (t, 7.2 Hz,
3H); 13C NMR δ 166.3 (d, 4.9 Hz), 132.1, 126.6, 123.2, 53.4 (d, J )
149.1 Hz), 50.1 (d, J ) 150.2 Hz), 38.0, 37.6, 29.5, 26.8, 18.6, 17.0,
13.5, 13.3; LSIMS-MS(-) 270.0 m/z (MH- - HCl).
Inhibition by Inactive Substrates. These inhibition experiments
were performed by obtaining the steady-state kinetic constants, in an
analogous manner as described above, in the presence of fixed
concentrations of inactive substrates. The norleucine substrate, 5, was
used in all of the inhibition experiments. In these experiments, the
DMSO/substrate stock solutions contain a fixed amount of inactive
substrate. The approximate inhibition constant was obtained from the
equation KMapp ) KMo(1 + [I]/Ki), where KMapp was the experimental

1438 J. Am. Chem. Soc., Vol. 121, No. 7, 1999
Wade and Scanlan
Table 2. Inhibition Constants of the Phosphonatesa
Table 1. Steady-State Kinetic Analysis of Transition State Binding
of the Homologous Seriesa
kcat
KM
(mM)
kcat/KM
∆∆Gb
(kcal/mol)
substrate
(s-1
)
(s-1‚M-1
)
1 -H
no catalysis
1.0 ( 0.1
2 -CH3
3.4 ( 0.4
290 ( 40
180 ( 40
+2.2
+2.5
+0.6
0.0
3 -CH2CH3
4 -CH2CH2CH3
5 -CH2(CH2)2CH3 2.1 ( 0.1
6 -CH2(CH2)3CH3 1.3 ( 0.1
7 -CH2(CH2)4CH3 no catalysis
0.37 ( 0.03 2.1 ( 0.5
1.4 ( 0.1
0.32 ( 0.04 4500 ( 60
0.18 ( 0.03 12000 ( 1400
0.88 ( 0.12 1500 ( 20
+1.2
aAll reactions were performed in 50 mM borate, 150 mM NaCl (pH
8.70) at 24.5 ( 1.0 °C. The kinetic constants were determined from
saturation plots of initial rates versus substrate concentration. Substrate
concentrations used are listed in the Experimental Section. The errors
shown were obtained from calculated fits (KaleidaGraph-Synergy
Software) of the data to the Michaelis-Menton equation. The errors
in the kcat/KM values were from the propagation of the fitted kcat and
K
M errors.80 All of the catalyzed reactions were inhibited to background
levels with saturating concentrations of 5p (>25 mM). The ∆∆Gb
values were calculated from the equation -RT ln[(kcat/KM)X/(kcat/KM)5]
where R is the gas constant and T is the absolute temperature. X
corresponds to a substrate to which 5 is being compared. The detection
limit that has been estimated for the 17E8 at pH 8.70 is approximately
5-10 M-1 s-1 for kcat/KM. This estimate is based on the lowest kcat
value detected (3) and the highest substrate concentration used, 44 mM
(1). We believe this to represent an upper detection limit as the kcat
value for 3 could be obtained easily and reproducibly.
KM obtained in the presence of inactive substrate and KMo is the KM
obtained in the absence of the inactive substrate. In these experiments
it was assumed that the inactive substrates were competitive inhibitors.
This assumption is substantiated by the fact the there was no change
in kcat for norleucine in the presence of these inhibitors.18
a The constants were determined by plotting the slope values obtained
from Lineweaver-Burk plots against inhibitor concentration and
obtaining the intercept on the [inhibitor] axis which yields -Ki. The
slope values are equal to ((KM(1 + [I]/Ki)/Vmax). The inhibitor
concentrations used to obtain the Ki values are given in the Experimental
Section. The ∆∆Gb values were calculated from the equation -RT
ln[(Ki)X/(Ki)5p]. The Ki values were treated as thermodynamic equilib-
rium binding constants as the nature of inhibition was purely competive
(with respect to 5) of the 17E8-catalyzed reaction (data not shown).18
Results
Homologous Series. To investigate the use of binding energy
for catalysis between the methylene units of the side chain and
the recognition pocket of 17E8, we used a series of phenyl ester
substrates and phosphonates that contain aliphatic side chains
of varying lengths (Figure 2, 1-7). Table 1 shows the values
of kcat, KM, kcat/KM, and the relative transition state stabilization
energies, ∆∆Gb (relative to 5) for the series of substrates. Both
kcat and KM values were effected by changing the number of
methylene groups in the side chain. The magnitude of these
effects varied with respect to each of the steady-state constants.
The changes in kcat were the smallest of the three values,
spanning a range of about 5-fold (0.4-2.1 s-1). The changes
in KM (0.18-3.4 mM) and kcat/KM (180-12000 s-1 M-1) are
largersspanning almost 2 orders of magnitude. The values of
kcat and kcat/KM were the largest for 5 and decreased in the order
n-propyl 4 ≈ n-pentyl 6 > methyl 2 ≈ ethyl 3. Removing a
methylene group from the side chain decreased ∆∆Gb by as
much as 1.9 kcal/mol and as little as 0.3 kcal/mol depending
on the position in the side chain. The substrate with no side
chain, 1, showed no detectable activity, as did the substrate with
an n-hexyl side chain, 7. In addition, neither of these substrates
competed effectively with hydrolysis of 5, as there was no
change in the kcat or KM values (data not shown), at high
concentrations (44 mM, 1, and 3 mM, 7).
active site of the antibody and that the Ki values represent the
thermodynamic binding constants Kd for each phosphonate.18
The Ki values for this series ranged from 1.7 mM to 470 nM,
spanning about 4 orders of magnitude. The order of increasing
affinity of the phosphonates to 17E8 mirrors that of the catalytic
specificity of the corresponding substrates. The energetic
changes, ∆∆Gb, reflected by the Ki values for the side chain
variation are similar to those for the kcat/KM values for the
corresponding substrates.
Two phosphonates (1p and 11p) were used to probe transition
state binding upon complete removal of the side chain. The
substrates corresponding to both of the phosphonates are not
cleaved by the antibody.19 The Ki values of 1p and 11p are 1.7
mM (∆∆Gb ) 4.8 kcal/mol) and 53 µM (2.8 kcal/mol),
respectively (Table 2). 1p is an obvious candidate because, like
the rest the homologous series, it serves as a probe for the
methylene-pocket interactions without changing other substit-
uents on the molecule. However, the absence of a substituent
on the R-carbon introduces the additional complexity of
conformational freedom. Thus, the ∆∆Gb for 1p reflects both
the lack of side chain-pocket interactions and the newly
introduced rotational entropic requirement for binding the
phosphonate.20,21 11p also serves as a probe for investigating
the effects of removing the side chain. In this molecule, extra
Similarly, the Ki values of the corresponding phosphonates
(1p-6p, Figure 3) were affected by the changes in the number
of methylene units in the side chain. The Ki values for the
phosphonates are shown in Table 2. All of the phosphonates
were competitive inhibitors of the catalyzed hydrolysis of
substrate 5 (data not shown), suggesting that they bind in the
(19) The substrate corresponding to 1p is 1. The hydrolysis of phenyl
acetate, the substrate that corresponds to the phosphonate, 11p, was not
catalyzed by 17E8 (data not shown).
(20) Ramakrishnan, C.; Ramachandran, G. N. Biophys. J. 1965, 5, 909-
933.
(18) Segel, I. H. Enzyme Kinetics; John Wiley and Sons: New York,
1975; p 957.
(21) Ramachandran, G. N.; Sasisekharan, V. AdV. Protein Chem. 1968,
23, 283-437.

Remote Binding Energy in Antibody Catalysis
J. Am. Chem. Soc., Vol. 121, No. 7, 1999 1439
Table 3. Steady-State Kinetic Analysis of Transition State Binding
of the Branched Series [for accompanying notes see Table 1]
Figure 4. Bound side chain conformation. (Left) Phosphonate hapten
in bound conformation. Coordinates are taken from 17E8‚hapten PDB
file 1eap. (Right) Newman projection of the n-butyl side chain (Câ-
Cꢀ). The hapten side chain dihedral angle in (a) bound low-energy
eclipsed conformations and (b) low-energy anti conformations.
conformational freedom has not been introduced due to the
degenerate conformers upon rotation about the R-C-P bond;
however, the interactions between the n-formyl group and the
protein are lost. Although these molecules were used to
investigate the same problem, they have very different binding
affinities. The ∆∆Gb values (relative to 5p) nevertheless indicate
that the side chain is very important for phosphonate binding.
The anomolous tight binding of 11p could be a result of
restriction of conformation due to the bond deletion, the removal
of an unfavorable interaction between 17E8 and the n-formyl
group, or changes in the solvation properties of the molecule.
Branched Series. We used substrates which were branched
at different positions to determine steric and geometric restric-
tions that govern the interactions between the substrate side
chain and the recognition pocket. The kinetic values for these
substrates are shown in Table 3. The γ-branched leucine ester
(11) was a substrate for the antibody, whereas the R- (8) and
â-branched (9, 10) substrates were not hydrolyzed by the
antibody. The kcat value of 2.1 s-1 for the leucine substrate (11)
was similar to that of its linear counterpart 5, whereas the KM
value is10-fold higher (1.6 mM), resulting in a decreased kcat/
KM (11 compared to 5). The presence of high concentrations of
9 (4 mM) and 10 (2 mM) approximately doubled the KM value
of 5 (relative to the value in their absencesdata not shown)
with no change in kcat. These results yield the approximate Ki
values of 4 mM for 9 and 10. The presence of a high
concentration of 8 did not change the KM (or kcat) value of 5.
The Ki values of phosphonates that contain branched side
chains (7p-10p) were also obtained (Table 2). As with the
homologous phosphonate series, these were found to be
competitive inhibitors of the catalyzed reaction. Phosphonate
9p, which corresponds to the γ-branched substrate 11, had a Ki
value of 18 µM (∆∆Gb ) 2.2 kcal/mol). The â-branched
phosphonate, 7p, was a respectable binder to the antibody
binding site with a Ki value of 68 µM, whereas the correspond-
ing substrate 9 was not hydrolyzed by 17E8. Its Ki value is
similar to that of 2p, whose corresponding substrate was cleaved
by the antibody. We did not use a substrate that corresponded
to phosphonate 10p, but this molecule was of interest because
it contained a bulky side chain which would presumably require
large structural changes in the recognition pocket for binding.
The Ki of 1.1 mM is much larger than those of the homologous
series. The energetic changes for binding these phosphonates,
7p and 10p, relative to the phosphonate 5p are 2.4 and 4.0 kcal/
mol, respectively.
Table 4. Steady-State Kinetic Analysis of Transition State Binding
of the Conformationally Restricted Series [for accompanying notes
see Table 1]a
a The prepared substrate was determined to contain a 50:50 mixture
of cis and trans isomers (1H NMR). The values in parentheses are
corrected for the actual concentration of the cis isomer. The trans isomer
has been shown to be inactive as a substrate.
the dihedral defined by the γ and δ carbons (Figure 4). The
substrate containing the side chain with the cis double bond 13
was an active substrate whereas that containing the trans double
bond 12 was not hydrolyzed (Table 4).22 There is a small
decrease in the kcat value of 13, relative to that of the linear
substrate 5 (2.1 to 1.4 s-1) and essentially no change in KM
(0.18 to 0.21 mM); the kcat/KM value was decreased only 2-fold
(12000 to 6800 M-1 s-1). The presence of a high concentration
of 12 (2.7 mM) approximately tripled the KM value of 5 relative
to its absence (data not shown) with no change in kcat. This
result yields an approximate Ki of about 1.6 mM for 12. The
substrate with the alkynyl side chain, 14, was also processed
by the antibody. Its kcat and KM values are higher than those of
the parent substrate 5. The increased kcat may be solely due to
an electronic effect, since the buffer-catalyzed reaction of 14 is
increased over that of 5 to the same extent.
Discussion
Nature has evolved many enzymes to use their specificity
pockets efficiently. In addition to overall transition state
stabilization, these pockets have been experimentally shown to
serve several specific roles for catalysis.23-27 One role is to assist
in the formation of a productive Michaelis complex with the
(22) The substrate 13 was prepared as a 1:1 mixture of the cis and trans
isomers whereas 12 was purely trans.
(23) Knowles, J. R. J. Theor. Biol. 1965, 9, 213-228.
(24) Hedstrom, L.; Farr-Jones, S.; Kettner, C. A.; Rutter, W. J.
Biochemistry 1994, 33, 8764-8769.
Conformationally Restricted Substrate Series. The con-
formationally restricted series was used to investigate the effects
of side chain conformation on catalytic activity. The crystal
structure of the 17E8‚hapten complex suggests the binding of
a high-energy hapten side chain rotamer conformation about
(25) Bone, R.; Frank, D.; Kettner, C. A.; Agard, D. A. Biochemistry
1989, 28, 7600-7609.
(26) Perona, J. J.; Craik, C. S. Protein Sci. 1995, 4, 337-360.
(27) Thompson, R. C. Biochemistry 1974, 13, 5495-5501.

1440 J. Am. Chem. Soc., Vol. 121, No. 7, 1999
Wade and Scanlan
Interestingly, the range of Ki values spanned in the 17E8 system
is comparable to ranges seen with thermolysin and R-lytic
protease.25,37
The parameters that govern specificity with 17E8 are also
similar to those that dictate specificity in enzymatic systemss
substrate side chain size, shape, and geometry. The importance
of size is best exemplified by the homologous series results
which indicate that there is an optimal length for a linear side
chain of four carbons. The addition or subtraction of methylene
groups to this optimal side chain results in a loss of transition
state stabilization. The results with the branched substrates
suggest that a linear side chain structure is not required, but
only the γ-branching geometry is allowed. This tolerance for
only certain side chain branching is also observed with several
serine proteases as γ-branching is allowed and â-branching is
very detrimental.38,39
A strict conformational requirement to form catalytically
productive side chain-pocket interactions is illustrated by the
results from the conformationally restricted series in which the
side chain with the cis double bond (13) was a substrate and
the side chain with the trans double bond (12) was not. The
torsional angle (Câ-Cꢀ) measured from the refined crystal-
lographic coordinates is approximately 124° (Figure 4). The
lowest energy conformation for this torsion angle is 180°.40-42
However, in crystallographic models, dihedral angles are
difficult to define unambiguously within more than about 20°,
especially in cases where the dihedral in question extends
beyond the Câ-Cγ bond.43-45 The kinetic results suggest that
the bound conformer of the four-carbon side chain of 5
resembles a high-energy rotamer more closely mimicked by the
side chain of 13 and that the conformer mimicked by 12 is one
that cannot bind to the antibody.46
Figure 5. Transition state analogue binding versus catalytic efficiency.
ln Ki vs ln kcat/KM (slope ) -1.13 ( 0.17; R ) 0.95). The data were
fit with the KaleidaGraph curve-fitting program (data taken from Tables
1, 3, and 4). The inhibition data for the methionine phosphonate will
be reported in a future paper.
correct substrates and to hinder the formation of incorrect
substrates. A second role is to help precisely position the
substrate reactive groups relative to those on other substrates
or catalytic groups in the enzyme active site. A third role of
specificity pockets is to assist in the preferential stabilization
of transition states and high-energy intermediates. Although
these are separate roles, nature has coupled them to optimize
the efficiency of these interactions in catalysis of specific
substrates.26,28-31 In this study, we investigate how binding
energy in a specificity pocket programmed by hapten design is
used in the mechanism of an esterolytic antibodysa protein that
has not been optimized by nature to perform catalysis.
Overall Transition State Stabilization. Alternative substrate
and transition state analogues provide two different probes to
investigate how 17E8’s specificity pocket interactions affect the
energy of the transition state. There is a good linear correlation
between the ln(Ki) values of the phosphonates and the ln(kcat/
KM) values of the corresponding substrates, indicating the
presence of similar binding interactions between 17E8 and both
the phosphonate inhibitors and the transition states of the
catalyzed reactions (Figure 5), suggesting that the inhibitors are
good approximations of the transition state.32,33 The analogues
contain sufficiently different side chains; thus, the correlation
implies that the free energy modulation of the transition state
complexes is a result of changes in the side chain-pocket
interactions and not a result of changes in the catalytic
mechanism with the different substrates.
Another parameter that has been shown to affect specificity
in analogous enzyme systems is side chain hydrophobicity. The
log(Ki) and log(kcat/KM) values of the phosphonates and
substrates correlate with the hydrophobicity parameter, π (Figure
6, see the caption for the definition of π) which suggests that
the hydrophobic effect may provide a large contribution to
(37) Bartlett, P. A.; Marlowe, C. K. Biochemistry 1983, 22, 4618-4624.
(38) Bigler, T. L.; Lu, W.; Park, S. J.; Tashiro, M.; Wieczorek, M.; Wynn,
R.; Michael Laskowski, J. Protein Sci. 1993, 2, 786-799.
(39) Lu, W.; Apostol, I.; Qasim, M. A.; Warne, N.; Wynn, R.; Zhang,
W. L.; Anderson, S.; Chiang, Y. W.; Ogin, E.; Rothberg, I.; Ryan, K.;
Laskowski, M. J. Mol. Biol. 1997, 266, 441-461.
(40) Compton, D. A. C.; Montero, S.; Murphy, W. F. J. Phys. Chem.
1980, 84, 3587-3591.
(41) Allinger, N. L.; Yuh, Y. H.; Lii, J.-H. J. Am. Chem. Soc. 1989,
111, 8551-8559.
The 17E8 specificity profile resembles that of natural enzymes
that contain similar specificity pockets (e.g., chymotrypsin and
methionyl-tRNA synthetase).34-36 This result is gratifying as
this is the first catalytic antibody specificity pocket to be
systematically studied. The 2 orders of magnitude range spanned
by the substrates’ kcat/KM values is small compared to those of
similar substrates with chymotrypsin (4 orders of magnitude)
and methionyl-tRNA synthetase (7 orders of magnitude).
(42) The energetic difference (∆H) between the gauche and anti
conformations of n-butane is 0.7-1.2 kcal/mol, with the anti conformer
being preferred. The energetic differences (∆H) between the two eclipsed
forms and the anti conformation are 3.5 kcal/mol (H/CH3) and >4.5 kcal/
mol (CH3/CH3), respectively (see refs 40 and 41).
(43) Janin, J.; Wodak, S.; Levitt, M.; Maigret, B. J. Mol. Biol. 1978,
125, 357-386.
(44) Pickett, S. D.; Sternberg, M. J. E. J. Mol. Biol. 1993, 231, 825-
839.
(45) Schrauber, H.; Eisenhaber, F.; Argos, P. J. Mol. Biol. 1993, 230,
592-612.
(28) Gron, H.; Breddam, K. Biochemistry 1992, 31, 8967-8971.
(29) Bone, R.; Shenvi, A. B.; Kettner, C. A.; Agard, D. A. Biochemistry
1987, 26, 7609-7614.
(30) Jencks, W. P. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4046-4050.
(31) Kraut, J. Annu. ReV. Biochem. 1977, 46, 331-358.
(32) Wolfenden, R. Annu. ReV. Biophys. Bioeng. 1976, 5, 271-306.
(33) Transition states of biochemical processes; Gandour, R. D., Scho-
wen, R. L., Eds.; Plenum Press: New York, 1978.
(46) However, the energetic cost needed for the antibody to constrain
the side chain is not realized with the fixed rotamer mimick (13). This
conformer is about 3.5 kcal/mol higher in energy than the staggered, low-
energy conformer (see refs 40 and 41). Thus, structurally constraining the
side chain should allow for this energy to be expressed in greater transition
state stabilization. The decrease in activity (Tables 1 and 3) for 13 compared
to 5 is most likely a result of the fact that the cis isomer does not resemble
the bound rotamer precisely due to changes in the hybridization at the δ
and γ carbons as well as differences in the dihedral angle between 13 and
the actual bound rotamer. The alkene side chain is also less hydrophobic
(than 5), thus raising the desolvation cost for binding. The fact that 14 was
also a substrate suggests that the rotameric requirement is only important
for the torsion angle defined by carbons Cb-Ce.
(34) Dorovska, V. N.; Varfolomeyev, S. D.; Kazanskaya, N. F.; Klyosov,
A. A.; Martinek, K. FEBS Lett. 1972, 23, 122-124.
(35) Berezin, I. V.; Kazanskaya, N. F.; Klyosov, A. A.; Martinek, K.
FEBS Lett. 1971, 15, 125-128.
(36) Fersht, A. R.; Dingwall, C. Biochemistry 1979, 18, 1250-1255.

Remote Binding Energy in Antibody Catalysis
J. Am. Chem. Soc., Vol. 121, No. 7, 1999 1441
Figure 7. The top view of a space-filling model of the 17E8 (black)‚
5p (light and dark grey) complex. The model was generated with the
MIDAS program.75,79
17E8’s active site apparent hydrophobicity results in an
average free energy contribution to transition state stabilization
per methylene group of about 0.7 kcal/mol. This value is
essentially the same as the 0.5 kcal/mol value from experiments
in which small molecules were partitioned between the aqueous
and organic phases.50,51 The negligible difference between the
two values does not reflect the expected tighter methylene-
pocket interactions in the protein‚ligand complexes compared
to methylene-organic solvent interactions and the advantage
of having a preformed pocket.6,52 The ∆∆Gb per methylene
group is also significantly smaller than the corresponding values
in natural enzymes that contain similar side chain recognition
pockets that have evolved to form efficient binding interac-
tions.4-6,53 For the enzymatic systems, the ∆∆Gb per methylene
group determined from substrate homologous series experiments
range from 1.5 to 3.5 kcal/mol. The smaller average value for
∆∆Gb per methylene group indicates that 17E8 does not use
binding interactions as efficiently as an evolved enzyme that
contains a similar amino acid side chain recognition pocket.
The P1 side chain is completely buried upon the formation
6,54-56
of the transition state in serine proteases.
In contrast,
Figure 6. Specificity pocket hydrophobicity. Hydrophobicity (π) of
the substrate and phosphonate side chains versus the substrate steady-
state kinetic constants and phosphonate inhibition constants where π
) log(P/Po) where Po is the ratio of the solubility of the parent
compound (H-X) in an organic phase to that in the aqueous phase,
and P is that of a compound on which the H substituent has been
exchanged for R, to give R-X.77,78 (a) ln kcat/KM (slope ) 1.1 ( 0.4;
R ) 0.87). (b) ln Ki (slope ) -1.4 ( 0.2; R ) 0.98). (c) ln KM (slope
-0.82 ( 0.20; R ) 0.93).
partial solvent accessibility of the hapten side chain is clearly
evident in 17E8 (Figures 1b and 7). It has been shown that the
partial solvent accessibility of methylene groups in hydrophobic
cores results in a reduced contribution to protein stability
compared to those that are contained completely in cores.57,58
The reduced contribution results from a reduction in the
hydrophobic effect and the number of packing interactions. An
(49) Although hydrophobicity may play a major role, there are other
factors that are also expected to be major contributors to substrate and
transition state binding including statistical entropic factors and enthalpic
contributors from the formation of van der Waals packing interactions.
Sneddon, F. S.; Tobias, D. J. Biochemistry 1992, 31, 2842-2846 and
Morton, A.; Baase, W. A.; Mathews, B. W. Biochemistry 1995, 34, 8564-
8575.
transition state binding.47 This result is supported by the crystal
structure of the 17E8‚5p complex as the majority of atoms
within 5 Å of the side chain carbons are from hydrophobic and
aromatic amino acid residues (see Figure 1a).9 In studies with
chymotrypsin, the log(kcat/KM) values of a series of substrates
similar to ours correlated linearly with π as did another serine
protease subtilisin which shares a specificity similar to that of
chymotrypsin.34,35,48 The slopes of the log(kcat/KM) vs hydro-
phobicity obtained from these enzymatic systems are larger than
those obtained from 17E8.49
(50) Rose, G. D.; Wolfenden, R. Annu. ReV. Biophys. Biomol. Struct.
1993, 22, 381-415.
(51) Pace, C. N. In Methods in Enzymology; 1st ed.; Johnson, M. L.,
Ackers, G. K., Eds.; Academic Press: San Diego, 1995; Vol. 259; p 761.
(52) Richards, F. M. Annu. ReV. Biophys. Bioeng. 1977, 6, 151-176.
(53) Fersht, A. R.; Shindler, J. S.; Tsui, W.-C. Biochemistry 1980, 19,
5520-5524.
(54) Perona, J. J.; Hedstrom, L.; Rutter, W. J.; Fletterick, R. J.
Biochemistry 1995, 34, 1489-1499.
(47) The substrates 6 and 11 (and phosphonates 6p and 11p) were not
included in the correlations because we believe that these molecules could
not be accommodated without changes in pocket structure or binding mode,
making the analysis of these compounds ambiguous.
(48) Estell, D. A.; Graycar, T. P.; Miller, J. V.; Powers, D. B.; Burnier,
J. P.; Ng, P. G.; Wells, J. A. Science 1986, 233, 659.
(55) Bode, W.; Huber, R. Eur. J. Biochem. 1992, 204, 433-451.
(56) Stroud, R. M. Am. Sci. 1974, 231, 74-88.
(57) Jackson, S. E.; Moracci, M.; elMasry, N.; Johnson, C. M.; Fersht,
A. R. Biochemistry 1993, 32, 11259-11269.
(58) Otzen, D. E.; Rhiennecker, M.; Fersht, A. R. Biochemistry 1995,
34, 13051-13058.

1442 J. Am. Chem. Soc., Vol. 121, No. 7, 1999
Wade and Scanlan
extension of this can similarly be drawn to the carbons in the
side chain of 5p and most likely contributes to the apparent
unoptimized nature of the 17E8 recogniton pocket.
Formation of the Michaelis Complex. 17E8 does use the
free energy from the side chain-pocket interactions to stabilize
the Michaelis complex. This use of binding energy is especially
apparent for the homologous series substrates in which the
deletion of each methylene group from 5 (to 2) results in an
increase in KM. As with the transition state, side chain size,
geometry, conformation, and hydrophobicity (Figure 6) affect
the free energy of the 17E8 Michaelis complex, lending further
support for the participation of the pocket for substrate binding.
The KM increase for the substrates 6, 11, and 14 further
demonstrates this sensitivity.
The lack of detectable catalysis for 1, 7-10, and 12 does
not rigorously prove that the substrates cannot form ground-
state Michaelis-type complexes with 17E8. To determine the
free energies of their Michaelis-type complexes, the approximate
Ki values of these compounds were obtained. The free energies
for the 17E8 complexes with 9, 10, and 12 are 1.4-2 kcal/mol
greater than those for the 17E8‚5 complex. The Ki values of 1,
7, and 8 could not be obtained because of the solubility limits
of the compounds; thus, the free energies corresponding to the
Ki values of these compounds must be greater even than those
determined. These results lend further support to the side chain-
pocket interactions being important for the free energy of the
Michaelis complex. Destabilizing interactions are introduced in
the case of the long (7) and branched (8-10) substrates, whereas
many of the favorable interactions are removed with the
substrate 1.
Figure 8. Active site interactions of serine proteases and 17E8. (a)
R-Lytic protease complexed with methoxysuccinyl-A-A-P-boroAla.25
Coordinates for the complex were taken from the PDB file lp02. (b)
17E8‚5p complex. Active site hydrogen bonds are indicated by the
arrows. The figures were generated with the MIDAS program.75
Alignment of Catalytic Groups. The side chain-pocket
interactions are used to assist in the alignment of the oxyanionic
intermediate to the stabilizing cationic center on 17E8. Interest-
ingly, the results from the homologous series indicate that only
one methyl group (substrate 2) is needed for proper alignment
of the substrate to the 17E8 catalytic machinery. The kcat values
do not increase significantly with the addition of larger side
chain groups (from 2 to 6). The presence of very short (1), long
(7), R- and â-branched (8-10), or conformationally incorrect
(12) side chains results in a loss of detectable catalysis.
mol, respectively, relative to 5p) indicate the stability of these
complexes has somehow been compromised. The anomolous
tight binding of transition state analogues that correspond to
substrates that are not turned over or slowly turned over has
also been observed and structurally characterized for R-lytic
protease.25 The ability to bury more hydrophobic surface area
at the expense of several active side hydrogen bonds is
postulated to be one of the reasons for the anomolous binding
behavior and loss of catalysis. The prevalence of side chain-
pocket interactions in phosphonate, and presumably transition
state, binding is also demonstrated with 10p.
In the serine protease systems, additional subsite binding,
active site hydrogen bonds, and the oxyanion hole are used in
addition to the interactions in the S1 pocket to obtain the large
rate accelerations (Figure 8a).29,54,59 Interestingly, this strategy
is used by most proteases regardless of the overall tertiary
protein structure.55 The energy gained from the noncovalent
interactions in the specificity pocket is a function of all of these
components, some of which are remote from the specificity
pocket.6,24,60-63 By coupling the S1-P1 interactions to other
machinery in the active site, the proteases have created a
cooperative network that increases the apparent energy of S1-
P1 interactions.30 17E8 does have a network of interactions that
can be used in concert with side chain-pocket interactions. The
17E8 active site contains a center for stabilizing the formation
One explanation for the lack of catalysis with these substrates
may be the inability of the transition states to interact produc-
tively with the catalytic center. The high Ki values of the inactive
substrates cannot fully account for the lack of detectable
catalysis as there are active substrates that have similarly high
KM values. To understand how substrates are discriminated
against in the transition state, the binding of transition state
analogues (phosphonates) corresponding to the several inactive
substrates was investigated. The weak binding of 1p, and
transition state binding, in itself provides a rationale of why
the corresponding substrate, 1, was not detectably hydrolyzed
by 17E8. The transition state analogue, 7p, is a respectable
binder (Kd similar to 2p), although the corresponding substrate,
9, was not cleaved detectably by 17E8. The ability of this
analogue to bind tightly may be due to its potential to bury
more hydrophobic surface area (80 Å2, 7p, vs 33 Å2, 2p). This
side chain burial must be at the expense of forming efficient
interaction with the oxyanion center as evident from the loss
of catalysis and the similar binding constant to 2p. The addition
of a methylene unit to the side chain of 7p results in an increase
in binding affinity (8p, 101 Å2), supporting the idea that the
tight binding of 7p partly results from the burial of hydrophobic
surface area. Nevertheless, the decreases in favorable free energy
of the complexes of 7p and 8p with 17E8 (+2.9 and +1.6 kcal/
(59) Hedstrom, L.; Szilagyi, L.; Rutter, W. J. Science 1992, 255, 1249-
1253.
(60) Berti, P. J.; Fearman, C. H.; Storer, A. C. Biochemistry 1991, 30,
1394-1402.
(61) Ingles, D. W.; Knowles, J. R. Biochem. J. 1967, 104, 369-377.
(62) Ingles, D. W.; Knowles, J. R. Biochem. J. 1968, 108, 561-569.
(63) Gron, H.; Meldal, M.; Breddam, K. Biochemistry 1992, 31, 6011-
6018.

Remote Binding Energy in Antibody Catalysis
J. Am. Chem. Soc., Vol. 121, No. 7, 1999 1443
of an oxyanion, but it does not use additional subsite interactions
to increase the apparent binding energies from the interactions
with oxyanion hole (Figure 8b). 17E8 is also missing active
site hydrogen bonds that serve as critical kinetic specificity
determinants that assist in the more productive use of the side
chain pocket binding energy.54,62
pocket interactions to increase kcat/KM and not to increase kcat
makes this clearly a case in which ground-state interactions are
inhibitory to increasing the turnover number.33
Conclusion
In this study, we find that 17E8 does use the binding energy
between the n-butyl side chain and its recognition pocket to
stabilize the bound transition state, indicating that catalytically
productive binding interactions that are remote from the catalytic
center can be programmed by hapten design. We also find that
this pocket has some characteristics similar to those contained
in evolved enzymes, including the mechanisms and molecular
forces used for specificity. One side chain methyl group is
sufficient to ensure high turnover and presumably alignment
of the carbonyl group on the ester substrate with the oxyanion
hole. Additional methylene groups increase the turnover number
almost negligibly but contribute substantially to the formation
of the Michaelis complex and catalytic efficiency (kcat/KM),
indicating that 17E8 uses these additional side chain-pocket
interactions uniformly in the Michaelis and transition state
complexes. This result substantiates the need for selection
techniques other than transition state analogue binding in
catalytic antibody screening procedures. Optimistically, this
suggests that this selection technique may provide a good
starting point for a catalyst and that further protein engineering
is needed for optimization. The 17E8 side chain-pocket
interactions are used less efficiently than those in enzymes as
the apparent binding energies gained from the methylene-
pocket interactions are significantly smaller in the 17E8 system
than those observed in enzymes. Analysis of the 17E8‚5p
complex reveals several possible sources of this reduced
apparent binding energy, including incomplete burial of the side
chain upon transition state formation and the absence of other
binding interactions that could be coupled to the side chain-
pocket to preclude wasteful side chain binding. These features
provide at least some insight into how 17E8 might be engi-
neered.
Preferential Stabilization of the Transition State. Although
overall transition state stabilization increases as methylene
groups are added to the side chain of substrates 1-5, the addition
of these units did not significantly increase the kcat values,
indicating that the binding energies from the additional meth-
ylene-pocket interactions were not used differentially between
binding the ground state Michaelis and the transition state
complexes. This uniform use of binding energy is thought to
be characteristic of an evolutionarily unoptimized catalyst.64-66
There are natural enzymes that use much of the binding energy
from side chain-pocket interactions for substrate binding
specificity and not transition state discrimination.11,36,53,67
However, in these systems, kinetic specificity determinants
(binding interactions that descriminate at the kcat level only) have
also been identifiedssuggesting that the uniform use of energy
from side chain-specificity pocket interactions is permissible
as long as there are other sources of differential binding.61,62,68-70
Because they are raised against transition state analogues,
and in most cases have been selected for tight binding to these
analogues, catalytic antibodies are usually designed to influence
only the E + Sq T (E‚S)q equilibriumsnot the equilibria E +
S T E‚S and E‚S T (E‚S)q. These generation techniques leave
no a priori reason for one to expect these proteins to enforce
the discriminatory use of remote binding energy between
antibody bound species. In fact, the general problem of product
inhibition with catalytic antibodies suggests that uniform binding
of common moieties on substrates, high-energy intermediates,
and products may be prevalent.2,71 This is in contrast to enzymes
which have evolved to use binding interactions selectively in
their mechansims and not at only one location on the reaction
coordinate.5,68,72-74 Indeed, a more optimized 17E8 might select
against Michaelis complexes that use too much side chain
binding energy. In the 17E8 system, the use of the methylene-
Acknowledgment. This study was supported by a grant from
the National Institutes of Health (GM 50672). T.S.S. is an Alfred
P. Sloan Research Fellow. H.W. is supported by a NSF
Predoctoral Fellowship. Mass spectral analyses of synthetic
compounds were provided by the UCSF Mass Spectrometry
Facility (A. L. Burlingame, Director) supported by the Biomedi-
cal Research Technology Program of the National Center for
Research Resources, NIH, NCRR, BRTP 01614, and NIH
NIEHS ES04705. We gratefully acknowledge D. A. Agard, S.
M. Miller, and J. Harris for critical review of the manuscript
and useful discussions.
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