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A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond
Author(s) -
DiMaio John,
Ni Feng,
Gibbs Bernard,
Konishi Yasuo
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80441-5
Subject(s) - scissile bond , chemistry , thrombin , stereochemistry , hirudin , tripeptide , binding site , active site , antithrombin , non competitive inhibition , peptide bond , amino acid , biochemistry , enzyme , biology , heparin , platelet , immunology
A synthetic hirudin peptide analog corresponding to N ′‐acetyl [D‐Phe 45 , ArgΨ(COCH 2 ) 47 , Gly 48 ]desulfo hirudin 55–65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow‐tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human α‐thrombin ( K i =3.7±0.3 × 10 −10 M) and bovine α‐thrombin (1.8±0.7 × 10 −9 M). P79 showed saturable inhibition of plasma APTT. The P 1 subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site‐directed tripeptide [D‐Phe‐Pro‐ArgΨ(COCH 2 )CH 3 COOCH 3 , P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α‐thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K 1 =17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α‐thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α‐thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N ???‐acetyl[D‐Phe 45 , ArgΨ(COCH 2 ) 47 , Gly 48 ]desulfo hirudin 45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH 2 ‐ and COOH‐terminal recognition sites.