Allosteric effects of a monoclonal antibody against thrombin exosite II

被引:31
作者
Colwell, NS
Blinder, MA
Tsiang, M
Gibbs, CS
Bock, PE
Tollefsen, DM
机构
[1] Washington Univ, Sch Med, Div Hematol, Dept Internal Med, St Louis, MO 63110 USA
[2] Washington Univ, Sch Med, Dept Pathol, St Louis, MO 63110 USA
[3] Gilead Sci Inc, Foster City, CA 94404 USA
[4] Vanderbilt Univ, Sch Med, Dept Pathol, Nashville, TN 37232 USA
关键词
D O I
10.1021/bi980925f
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We previously isolated a monoclonal antithrombin Ige 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 k(cat) for substrates with a P2 glycine residue but generally decreasing the k(cat) 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 hirudin(54-65). These results provide evidence for a direct allosteric linkage between exosite II and the catalytic site of thrombin.
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页码:15057 / 15065
页数:9
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