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References listed in PDB file
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Key reference
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Title
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Thermodynamic and structural basis for transition-State stabilization in antibody-Catalyzed hydrolysis.
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Authors
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M.Oda,
N.Ito,
T.Tsumuraya,
K.Suzuki,
M.Sakakura,
I.Fujii.
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Ref.
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J Mol Biol, 2007,
369,
198-209.
[DOI no: ]
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PubMed id
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Abstract
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Catalytic antibodies 6D9 and 9C10, which were induced by immunization with a
haptenic transition-state analog (TSA), catalyze the hydrolysis of a
nonbioactive chloramphenicol monoester derivative to generate a bioactive
chloramphenicol. These antibodies stabilize the transition state to catalyze the
hydrolysis reaction, strictly according to the theoretical relationship: for
6D9, k(cat)/k(uncat)=895 and K(S)/K(TSA)=900, and for 9C10, k(cat)/k(uncat)=56
and K(S)/K(TSA)=60. To elucidate the molecular basis of the antibody-catalyzed
reaction, the crystal structure of 6D9 was determined, and the binding
thermodynamics of 6D9 and 9C10 with both the substrate and the TSA were analyzed
using isothermal titration calorimetry. The crystal structure of the unliganded
6D9 Fab was determined at 2.25 A resolution and compared with that of the
TSA-liganded 6D9 Fab reported previously, showing that the TSA is bound into the
hydrophobic pocket of the antigen-combining site in an "induced fit"
manner, especially at the L1 and H3 CDR loops. Thermodynamic analyses showed
that 6D9 binds the substrate of the TSA with a positive DeltaS, differing from
general thermodynamic characteristics of antigen-antibody interactions. This
positive DeltaS could be due to the hydrophobic interactions between 6D9 and the
substrate or the TSA mediated by Trp H100i. The difference in DeltaG between
substrate and TSA-binding to 6D9 was larger than that to 9C10, which is in good
correlation with the larger k(cat) value of 6D9. Interestingly, the DeltaDeltaG
was mainly because of the DeltaDeltaH. The correlation between k(cat) and
DeltaDeltaH is suggestive of "enthalpic strain" leading to
destabilization of antibody-substrate complexes. Together with X-ray structural
analyses, the thermodynamic analyses suggest that upon binding the substrate,
the antibody alters the conformation of the ester moiety in the substrate from
the planar Z form to a thermodynamically unstable twisted conformation, followed
by conversion into the transition state. Enthalpic strain also contributes to
the transition-state stabilization by destabilizing the ground state, and its
degree is much larger for the more efficient catalytic antibody, 6D9.
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Figure 1.
Figure 1. Chemical transformation resulting from
antibody-catalyzed prodrug activation and chemical formulae of
the compounds used in this study. Antibodies 6D9 and 9C10 were
raised against chloramphenicol phosphonate 3 and catalyze the
hydrolysis of chloramphenicol ester 1 to generate
chloramphenicol 2 and the acid product. Sub-Lys (4) was used for
the ITC study.
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Figure 4.
Figure 4. Conformational changes of His L27d (a) and Trp
H100i (b) induced by TSA binding. The structures of 6D9 in the
unliganded form (sky blue) are superimposed on that of the
liganded form (blue), form II, re-refined based on the data from
the previous work.^10 Binding of the TSA “pulls” the L1 loop
through a hydrogen bond between the phosphate and the catalytic
His L27d. The indole ring of Trp H100i in the unliganded form
occupies a hydrophobic pocket, whereas it flips out to allow the
TSA to bind in the pocket in the complex. For clarity, the
hapten is represented as the transition-state analog 3.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
369,
198-209)
copyright 2007.
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Secondary reference #1
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Title
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Site-Directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization.
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Authors
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H.Miyashita,
T.Hara,
R.Tanimura,
S.Fukuyama,
C.Cagnon,
A.Kohara,
I.Fujii.
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Ref.
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J Mol Biol, 1997,
267,
1247-1257.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Antibody-catalyzed prodrug activation.
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Figure 2.
Figure 2. Restriction map of the expression vector pARA7
and outline of the construction of the antibody expression
plasmids. (a) The construction of the plasmid for the 6D9 Fab
expression. (b) Synthetic oligonucleotides for the plasmid
constructions. P, The promoter-operator region of ara B; S, the
synthetic signal sequence; T, the transcription terminator;
H[N], the amino-terminal sequence of the IgG heavy chain gene;
R, the ribosome binding site. Restriction sites are: N, NcoI; K,
KpnI; H, HindIII; Xh, XhoI; Sp, SpeI; Sc, SacI; Xb, XbaI.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Secondary reference #2
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Title
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Correlation between antigen-Combining-Site structures and functions within a panel of catalytic antibodies generated against a single transition state analog
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Authors
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I.Fujii,
F.Tanaka,
H.Miyashita,
R.Tanimura,
K.Kinoshita.
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Ref.
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j am chem soc, 1995,
117,
6199.
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Secondary reference #3
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Title
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Crystallization and preliminary X-Ray analysis: transition state complex of a chloramphenicol prodrug activation specific catalytic antibody
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Authors
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O.Kristensen,
H.Miyashita,
D.G.Vassylyev,
F.Tanaka,
I.Fujii,
K.Morikawa.
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Ref.
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protein and peptide letters, 1995,
1,
252.
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Secondary reference #4
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Title
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A common ancestry for multiple catalytic antibodies generated against a single transition-State analog.
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Authors
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H.Miyashita,
T.Hara,
R.Tamimura,
F.Tanaka,
M.Kikuchi,
I.Fujii.
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Ref.
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Proc Natl Acad Sci U S A, 1994,
91,
10757.
[DOI no: ]
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PubMed id
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Secondary reference #5
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Title
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A common ancestry for multiple catalytic antibodies generated against a single transition-State analog.
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Authors
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H.Miyashita,
T.Hara,
R.Tanimura,
F.Tanaka,
M.Kikuchi,
I.Fujii.
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Ref.
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Proc Natl Acad Sci U S A, 1994,
91,
6045-6049.
[DOI no: ]
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PubMed id
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Secondary reference #6
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Title
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Prodrug activation via catalytic antibodies.
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Authors
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H.Miyashita,
Y.Karaki,
M.Kikuchi,
I.Fujii.
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Ref.
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Proc Natl Acad Sci U S A, 1993,
90,
5337-5340.
[DOI no: ]
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PubMed id
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Secondary reference #7
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Title
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Thermodynamic and structural analyses of hydrolytic mechanism by catalytic antibodies
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Authors
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M.Oda,
N.Ito,
T.Tsumuraya,
K.Suzuki,
I.Fujii.
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Ref.
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TO BE PUBLISHED ...
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