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Thermodynamic and structural basis for transition-state stabilization in antibody-catalyzed hydrolysis.

Oda M, Ito N, Tsumuraya T, Suzuki K, Sakakura M, Fujii I
J Mol Biol 369 198-209 (2007)
Related entries: 2dqt, 2dtm

Cited: 7 times
EuropePMC logo PMID: 17428500

Abstract

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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  1. Toward high-resolution homology modeling of antibody Fv regions and application to antibody-antigen docking. Sivasubramanian A, Sircar A, Chaudhury S, Gray JJ. Proteins 74 497-514 (2009)
  2. Computational structural analysis of an anti-L-amino acid antibody and inversion of its stereoselectivity. Ranieri DI, Hofstetter H, Hofstetter O. J Sep Sci 32 1686-1695 (2009)


Reviews citing this publication (1)

  1. A survey of the year 2007 literature on applications of isothermal titration calorimetry. Bjelić S, Jelesarov I. J Mol Recognit 21 289-312 (2008)

Articles citing this publication (4)

  1. A novel molecular analysis of genes encoding catalytic antibodies. Le Minoux D, Mahendra A, Kaveri S, Limnios N, Friboulet A, Avalle B, Boquet D, Lacroix-Desmazes S, Padiolleau-Lefèvre S. Mol Immunol 50 160-168 (2012)
  2. Contribution of the trifluoroacetyl group in the thermodynamics of antigen-antibody binding. Oda M, Saito M, Tsumuraya T, Fujii I. J Mol Recognit 23 263-270 (2010)
  3. A structural insight into the molecular recognition of a (-)-Delta9-tetrahydrocannabinol and the development of a sensitive, one-step, homogeneous immunocomplex-based assay for its detection. Niemi MH, Turunen L, Pulli T, Nevanen TK, Höyhtyä M, Söderlund H, Rouvinen J, Takkinen K. J Mol Biol 400 803-814 (2010)
  4. Effects of substrate conformational strain on binding kinetics of catalytic antibodies. Oda M, Tsumuraya T, Fujii I. Biophys Physicobiol 13 135-138 (2016)


Related citations provided by authors (7)

  1. Site-directed mutagenesis of active site contact residues in a hydrolytic abzyme: evidence for an essential histidine involved in transition state stabilization.. Miyashita H, Hara T, Tanimura R, Fukuyama S, Cagnon C, Kohara A, Fujii I J Mol Biol 267 1247-57 (1997)
  2. Correlation between Antigen-Combining-Site Structures and Functions within a Panel of Catalytic Antibodies Generated against a Single Transition State Analog. Fujii I, Tanaka F, Miyashita H, Tanimura R, Kinoshita K J. Am. Chem. Soc. 117 6199-6209 (1995)
  3. Crystallization and Preliminary X-Ray Analysis: Transition State Complex of a Chloramphenicol Prodrug Activation Specific Catalytic Antibody. Kristensen O, Miyashita H, Vassylyev DG, Tanaka F, Fujii I, Morikawa K Protein Pept. Lett. 1 252-255 (1995)
  4. A common ancestry for multiple catalytic antibodies generated against a single transition-state analog.. Miyashita H, Hara T, Tamimura R, Tanaka F, Kikuchi M, Fujii I Proc Natl Acad Sci U S A 91 10757 (1994)
  5. A common ancestry for multiple catalytic antibodies generated against a single transition-state analog.. Miyashita H, Hara T, Tanimura R, Tanaka F, Kikuchi M, Fujii I Proc Natl Acad Sci U S A 91 6045-9 (1994)
  6. Prodrug activation via catalytic antibodies.. Miyashita H, Karaki Y, Kikuchi M, Fujii I Proc Natl Acad Sci U S A 90 5337-40 (1993)
  7. Thermodynamic and Structural Analyses of Hydrolytic Mechanism by Catalytic Antibodies. Oda M, Ito N, Tsumuraya T, Suzuki K, Fujii I To be published -

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