Allosteric effects of a monoclonal antibody against thrombin exosite II.

Colwell NS, Blinder MA, Tsiang M, Gibbs CS, Bock PE, Tollefsen DM
Biochemistry. 1998 37 (43): 15057-65

PMID: 9790668 · DOI:10.1021/bi980925f

We previously isolated a monoclonal antithrombin IgG from a patient with multiple myeloma [Colwell et al. (1997) Br. J. Haematol. 97, 219-226]. Using a panel of 55 surface mutants of recombinant thrombin, we now show that the epitope for the IgG most likely includes Arg-101, Arg-233, and Lys-236 in exosite II. The IgG affects the rate at which thrombin cleaves various peptide p-nitroanilide substrates with arginine in the P1 position, increasing the kcat for substrates with a P2 glycine residue but generally decreasing the kcat for substrates with a P2 proline. The allosteric effect of the IgG is altered by deletion of Pro-60b, Pro-60c, and Trp-60d from the 60-loop of thrombin, which lies between exosite II and the catalytic triad. The effect of the IgG, however, does not depend on the presence or absence of sodium ions, a known allosteric regulator of thrombin. The IgG does not affect the conformation of thrombin exosite I as determined by binding of a fluorescent derivative of hirudin54-65. These results provide evidence for a direct allosteric linkage between exosite II and the catalytic site of thrombin.

MeSH Terms (18)

Allosteric Regulation Animals Antibodies, Monoclonal Binding Sites Cations, Monovalent COS Cells Enzyme Activation Epitopes Hirudins Humans Immunoglobulin Fab Fragments Immunoglobulin G Models, Molecular Multiple Myeloma Peptide Fragments Recombinant Proteins Sodium Thrombin

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