Active-site mapping of bovine and human blood coagulation serine proteases using synthetic peptide 4-nitroanilide and thio ester substrates

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Abstract
A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA [NA = 4-nitroanilide, Z = benzyloxycarbonyl] and Z-AA-Phe-Arg-NA where AA = Ala, Asn, Glu, Lys, Phe, Pro or Ser were used to map the S3 subsite of several serine proteases involved in blood coagulation. The enzymes studied included bovine thrombin, factor IXa, factor Xa, factor XIa, human .beta.-factor XIIa (factor XIIa fragment) and activated bovine and human protein C. Kinetic constants (kcat, KM and kcat/KM) for the enzymatic hydrolysis of the substrates by each enzyme were determined and used to compare the relative reactivities of the individual enzymes. Most of the enzymes reacted with all the substrates, although a few showed considerable specificity. Human .beta.-factor XIIa showed the highest reactivity of all the coagulation proteases studied and was also very substrate specific (kcat/KM ranged over 470-fold). The best substrate was Z-Lys-Phe-Arg-NA with kcat/KM = 140,000 M-1 s-1. Activated bovine protein C (best substrate = Z-Ser-Phe-Arg-NA), factor Xa (best substrate = Z-Glu-Gly-Arg-NA), and thrombin (best substrate = Z-Lys-Gly-Arg-NA) were the group of enzymes that showed next highest reactivity toward the substrates. Activated bovine protein C, thrombin and factor Xa displayed relatively little substrate specificity. Activated human protein C (best substrate = Z-Ser-Phe-Arg-NA) and factor XIa (best substrate = Z-Glu-Gly-Arg-NA) are moderately reactive enzymes. Activated human protein C is an extremely specific enzyme since it has such a large range of kcat/KM values. Factor IXa, although very slow, hydrolyzed 10 of the 14 substrates with the best substrate having kcat/KM = 503 M-1 s-1. Human .beta.-factor XIIa (best substrate = Z-Phe-Arg-SBu-i) and both bovine (best substrate = Z-Arg-SBzl) and activated human protein C (Z-Phe-Arg-SBu-i) were studied with a set of amino acid and dipeptide thio ester substrates. The best thio ester substrates were 10- to 359-fold better substrates than the best 4-nitroanilide substrates. Comparison of bovine .alpha.-factor XIIa with human .beta.-factor XIIa and activated bovine with human protein C showed substantial differences both in reactivity and in subsite preferences. There were also significant similarities. The results should be useful for the future development of sensitive and specific assays for coagulation enzymes.