Allosteric effects of a monoclonal antibody against thrombin exosite II.
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.
Colwell
N S
NS
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Blinder
M A
MA
Tsiang
M
M
Gibbs
C S
CS
Bock
P E
PE
Tollefsen
D M
DM
D000494:Allosteric Regulation / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D019556:COS Cells; D002414:Cations, Monovalent; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D006629:Hirudins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism / Q000494:pharmacology; D007074:Immunoglobulin G / Q000378:metabolism; D008958:Models, Molecular; D009101:Multiple Myeloma / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012964:Sodium / Q000502:physiology; D013917:Thrombin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002414:Cations, Monovalent; D000939:Epitopes; D006629:Hirudins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D010446:Peptide Fragments; D011994:Recombinant Proteins; C091596:hirudin (54-65); D012964:Sodium; D013917:Thrombin