Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, plasma kallikrein, and trypsin with amino acid and peptide thioesters: development of new sensitive substrates

Abstract

The subsite specificities of bovine factor IXa, factor Xa, factor XIa, factor XIIa, thrombin, plasma kallikrein, and trypsin were mapped with amino acid, dipeptide, and longer peptide thioester substrates. Each substrate contained a P1 Arg residue. The P1' residues included thiol residues which are analogues of valine, leucine, and isoleucine, respectively, and the P2 residue included 12 representative amino acid residues. Longer substrates with the sequence at the antithrombin III reactive site and at the zymogen activation site of various coagulation factors were also studied. The enzymatic hydrolysis of the thioesters was measured in the presence of 4,4'-dithiodipyridine which provides a very sensitive assay for the free thiol. The thioesters were excellent substrates for the coagulation factors studied, and the kcat/Km values for the best thioester substrates were higher than those previously reported for most of these enzymes. Thrombin and plasma kallikrein were the most active of the coagulation factors toward the thioester substrates. The best substrate for thrombin was Z-Gly-Arg-SCH2C6H5, although substrates containing proline in the P2 position were also quite effective. Some of the better substrates for plasma kallikrein had a P2 Phe or Trp residue. Factor IXa was the least reactive of the coagulation factors and hydrolyzed only four of the dipeptide thioesters. Substrates with bulky hydrophobic groups such as Phe or Trp in the P2 position were the most reactive with factor IXa. Factor Xa hydrolyzed all the thioester substrates tested, the most reactive being Z-Gly-Arg-SCH2C6H5. This is consistent with the fact that glycine and arginine are present in the P2 and P1 positions, respectively, of the factor Xa sensitive bonds in prothrombin which is the physiological substrate for factor Xa. Bovine factor XIa showed the least amount of specificity of the various coagulation factors and was quite reactive toward all of the thioester substrates. The most sensitive substrate for this enzyme was also Z-Gly-Arg-SCH2C6H5. Factor XIIa preferred the dipeptide with a P2 Phe, although the simpler thioester Z-Arg-SCH2CH(CH3)2 was more reactive. Trypsin hydrolyzed all of the thioester substrates at a high rate and showed little substrate specificity. With all enzymes studied, extension of the thioester substrate beyond P2 or the P1' thiol leaving group did not lead to an improvement in hydrolysis. Due to their high kcat/Km values and the ease of detecting the thiol leaving group, thioester substrates should be extremely useful for future studies of coagulation proteases.

High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays

“…The data are consistent with the physiological substrates of plasma kallikrein: factor XII (Thr . These findings are also consistent with previous studies of plasma kallikrein specificity (3,(17)(18)(19)(20). In a panel of 20 coumarin substrates, Iwanaga et al found FIG.…”

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.…”

“…McRae et al [26] In the present study, we could not find any good substrates for factors IXa and VIIa. The sequences located close to the cleavage sites required for the activations of factors X 142-441 and IX [39, 451 were taken as a guideline for synthesizing substrates, that is, Boc-Val-Val-Arg-NH-Mec, Boc-Leu-ThrArg-NH-Mec, and Boc-Phe-Ser-Arg-NH-Mec.…”

“…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. This difference seems to be due to the rate-limiting step of the peptide and ester hydrolysis, that is, rate-limiting acylation for amides and rate-limiting deacylation for esters [34].…”

“…The substrate specificity of plasma kallikrein has been studied with peptide-p-nitroanilides [26] and peptide-NH-Mec [7]. Plasma kallikrein exhibits a very strict specificity towards a Phe residue at the P2 site [7,261.…”