Open AccessStudy of the Specificity of Thrombin with Tripeptidyl‐ p ‐nitroanilide Substrates
Author(s) -
POZSGAY Marianne,
CS. SZABÓ Gabriella,
ELÖDI Pál,
GÁSPÁR Rezsö,
BAJUSZ Sandor,
SIMONSSON Roger
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb06229.x
Subject(s) - enzyme kinetics , chemistry , substrate (aquarium) , hydrolysis , kinetic energy , stereochemistry , enzyme , active site , organic chemistry , biology , physics , ecology , quantum mechanics
The kinetic behaviour of human thrombin has been studied with 26 tripeptidyl‐ p ‐nitroanilide substrates protected at the N terminus and with 9 unprotected ones. By the regression analysis of experimcntally determined 1/ K m , k cat , and k cat / K m values the individual contribution of each side chain of the various substrates to the kinetic parameters was calculated. The contributions to the kinetic parameters of the best substrates provide information about the structure of the binding site. The interaction of subsites Sl and PI, which determines primary specificity, proved to be marginal on the basis of contribution values, though it depends upon this contact whether the substrate is hydrolyzed at all. At subsite Sz proline appeared to be favourable. Subsite S3 plays an important role in efficiency. The best parameters were obtained here with the D configurations of bulky amino acid residues. The aromatic protecting groups applied did not improve the properties of substrates. BZDPhe‐Pro‐Arg‐Nan was predicted by calculation to be better than the protected substrates assayed. The compound was synthesized and tested. Its experimentally determined i/ K m , 55.1 mM −1 , was in good agreement with 50.9 mM −1 found by calculation.