C O MMU N I C A T I O N S
studied with a substrate, i.e., the programmed reactant, homologous
to the hapten. Antibody modification, via laborious site directed
mutations, has rarely improved catalytic efficiency. (See refs 11
and 12 for exceptions). Modification of the substrate, which is
13
invariably much simpler, may afford improved catalytic behavior
and even, as seen here, significant enantiodifferentiation at a reaction
center remote from the chiral center. Analysis of the triangular
relationship beween haptens, antibodies, and substrates, where any
of the three is judiciously varied, may provide empirical results
and/or insights, allowing improved rational design of catalytic
antibodies.
Acknowledgment. We thank Mike Pique for use of Figure 1.
We are grateful to Dr. J. Perez for assistance with the LURE
Synchrotron and to Dr. R. Arad-Yellin for the CD spectra and her
advice. B.S.G. and L.J.D. thank the US-Israel Binational Science
Foundation (Grant No. 94-00397) for support. This work was also
supported in part by EU-TMR grant ERBFMXCT 98-0193.
Supporting Information Available: Table of kinetic parameters
for D2.3-catalyzed hydrolysis of 2, 6D, and 6L, experimental details
for the synthesis and characterization of 6D, 6L, 7D, and 7L,
crystallization, data collection and processing, structure determinations,
and refinements (PDB codes for the two complexes are, respectively,
1
KN4 and 1KN2), and a table of crystallographic data and refinement
statistics for D2.3-7D and -7L (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(
1) Eliel, E. L.; Wilen, S. H.; Mandler, L. N. Stereochemistry of Organic
Compounds; Wiley: New York, 1994.
Figure 2. Complexes of D2.3 with 7D and 7L. Carbon is in gray, oxygen
in red, nitrogen in blue, and phosphorus in yellow; hydrogen at the chiral
centers of 7D and 7L is in purple. The chiral center hydrogen atoms are
included for clarity; their positions are deduced from those of the other
substituents of the alanine R-carbon. (A) Schematic view of D2.3 residues
that interact with ligand 7L. Residue numbering is according to Kabat et
al. The Fab CR trace is in green (dark for the heavy chain and light for
the light chain) and the ligand is in white. Hydrogen bonds are drawn as
dotted lines. (B) Stereoviews of the structures of 7L (top, blue net) and 7D
(
(
2) E.g.: Jones, J. B.; DeSantis, G. Acc. Chem. Res. 1999, 32, 99-107.
3) Tawfik, D. S.; Green, B. S.; Chap, R.; Sela, M.; Eshhar, Z. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 373-377.
(4) Tawfik, D. S.; Lindner, A.; Chap, R.; Eshhar, Z.; Green, B. S. Eur. J.
Biochem. 1997, 244, 619-626.
(
5) Charbonnier, J.-B.; Golinelli-Pimpaneau, B.; Gigant, B.; Tawfik, D. S.;
Chap, R.; Schindler, D. S.; Kim, S.-H.; Green, B. S.; Eshhar, Z.; Knossow,
M. Science 1997, 275, 1140-1142.
6) Gigant, B.; Charbonnier, J.-B.; Eshhar, Z.; Green, B. S.; Knossow, M.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 7857-7861.
1
4
(
(bottom, green net) superimposed within nets representing the corresponding
(7) Gigant, B.; Charbonnier, J. B.; Eshhar, Z.; Green, B. S.; Knossow, M. J.
Mol. Biol. 1998, 284, 741-750.
(
electron densities (2Fobs - Fcalc maps) in D2.3 Fab. The maps are contoured
8) Another modification of substrates 1 and 2 has also been studied (to be
submitted for publication).
9) However, such phenomena were described by early immunochemistry
researchers. See, for example: Pressman, D.; Bryden, J. H.; Pauling, L.
J. Am. Chem. Soc. 1948, 70, 1352-1358. Thus, a number of antibodies,
elicited with achiral haptens, catalyze asymmetric synthesis; e.g.: Hilvert,
D. Top. Stereochem. 1999, 22, 83-135.
at the one standard deviation level. (C) Superimposed structures of 7D
7
15
(
green) and 7L (white) and 5 (magenta) in D2.3 Fab.
(
in” these two ligands, which now adjust to a minimum energy
conformation within these constraints.
(
10) See Supporting Information.
Refinement of the atomic coordinates for the structures of the
complexes of the D- and L-Ala isomers 7D and 7L in D2.3 provides
models for these haptens that fit the electron density (Figure 2B).
The energy difference between the two complexes, 0.5 kcal/mol
(
11) Baca, M.; Scanlan, T. S.; Stephenson, R. C.; Wells, J. A. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 10063-10068.
(12) Romesberg, F. E.; Spiller, B.; Schultz, P. G.; Stevens, R. C. Science 1998,
279, 1929-1933.
(13) (a) E.g., “substrate attenuation”: Janda, K. D.; Benkovic, S. J.; McLeod,
D. A.; Schloeder, D. M.; Lerner, R. A. Tetrahedron 1991, 47, 2503-
(deduced from the affinities of 7D and 7L for D2.3; see below),
2506. (b) Tawfik, D. S.; Zemel, R. R.; Arad-Yellin, R.; Green, B. S.;
cannot be ascribed to one dominant strong intermolecular interac-
tion; it is due to the sum of different interactions of the haptens
with the antibody, associated with slightly different positions of
these haptens in the complexes (Figure 2C), and to their different
conformational energies when locked therein. Although the differ-
ences in the structures of 7D and 7L in D2.3 are subtle, these
differences are manifested by consistent interactions and chemical
behavior. Thus, the affinity of 7D is 50 nM while that of 7L is 26
Eshhar, Z. Biochemistry 1990, 29, 9916-9921. (c) Wada, Y.; Yamamoto,
M.; Isamu, I.; Ono, M. Chemistry Lett. (Jpn.) 1997, 1223-1224. (d) The
hapten-antibody-substrate relationship is well-exemplified by the family
of antibodies and substrates used in conjunction with a pyridinium hapten.
Reymond, J.-L.; Janda, K. D.; Lerner, R. A. Angew. Chem., Int. Ed. Engl.
1
991, 30, 1711-1713 and subsequent papers. (e) A recent hapten
modification also illustrates this notion. Takahashi, N.; Kakinuma, H.;
Liu, L.; Nishi, Y.; Fujii, I. Nature Biotechnol. 2001, 19, 563-567.
14) Kabat, E. A.; Wu, T. T.; Perry, H. M.; Gottesman, K. S.; Foeller, C.
Sequences of Proteins of Immunological Interest, 5th ed.; National Institute
of Health: Bethesda, MD, 1991.
(
10
(15) Figures were drawn with Molscript (A, C), Bobscript (B) and Raster3D.
nM (measured by fluorescence quenching ) and the K
µM) is lower than that of 6D (45 µM).
M
of 6L (10
(
a) Kraulis, P. J. Appl. Crystallogr. 1991, 24, 924-950. (b) Esnouf, R.
M. J. Mol. Graph. 1997, 15, 133-138. (c) Merritt, E. A.; Bacon, D. J.
Methods Enzymol. 1997, 277, 505-524.
In catalytic antibody research, a single hapten is typically used
to generate a large number of catalytic antibodies which are then
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