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

Abstract

A series of 14 tripeptide 4-nitroanilide substrates of the type Z-AA-Gly-Arg-NA 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

“…2a). The P2-Gly was most effective as previously reported [7,24,26,35,361, resulting in the increase of the k,,, value. In addition, the Gly and Ala residues contributed to increase the l/Km value.…”

“…In contrast, the peptide-NH-Mec substrates with a Phe residue at the P2 site were less reactive. Stone and Hofsteenge [33] and Cho et al [24] also reported that a-thrombin cleaves poorly peptide-p-nitroanilide substrates with a Phe residue at the P2 site. On the other hand, McRae et al [26] have found that the P2-Phe derivative is a good substrate for a-thrombin, using peptide thioesters.…”

“…The specificity of this enzyme has been examined by using peptide-NH-Mec substrates [9], peptide p-nitroanilides [33], thioesters [24], and peptidechloromethane inhibitors [33]. The sensitive substrates so far known were as follows: Boc-Leu-Ser-Thr-Arg-NH-Mec with kc,,/Km = 25000 M-' s-' [9], Z-Ser-Phe-Arg-NH-Np with k,,,/Km = 65000 M-' s-' [24], and D-Ile-Pro-Arg-NH-Np with kc,,/Km = 330000 (for human activated protein C) [33]. The enzyme has been demonstrated to select an apolar residue such as Phe at the P2 site [24] and an apolar D-amino acid residue at the P3 site [33].…”

“…The sensitive substrates so far known were as follows: Boc-Leu-Ser-Thr-Arg-NH-Mec with kc,,/Km = 25000 M-' s-' [9], Z-Ser-Phe-Arg-NH-Np with k,,,/Km = 65000 M-' s-' [24], and D-Ile-Pro-Arg-NH-Np with kc,,/Km = 330000 (for human activated protein C) [33]. The enzyme has been demonstrated to select an apolar residue such as Phe at the P2 site [24] and an apolar D-amino acid residue at the P3 site [33]. Moreover, Ohno et al [9] and Cho et al [24] have indicated the importance of Ser at the P3 site for effective hydrolysis.…”

“…The enzyme has been demonstrated to select an apolar residue such as Phe at the P2 site [24] and an apolar D-amino acid residue at the P3 site [33]. Moreover, Ohno et al [9] and Cho et al [24] have indicated the importance of Ser at the P3 site for effective hydrolysis. Unlike factor Xa, activated protein C accommodates a bulky residue such as Val or Asp(OBz1) at the P2 and P3 sites (Supplement Fig.3a, b and c).…”