H-Ala-Phe-Pro-pNA

Serine proteases catalyze the hydrolysis of amide bonds of their protein and peptide substrates through a mechanism involving the intermediacy of an acyl-enzyme. While the rate constant for formation of this intermediate, k(2), shows a dramatic dependence on peptide chain length, the rate constant for the intermediate's hydrolysis is relatively insensitive to chain length. To probe the mechanistic origins of this phenomenon, we determined temperature dependencies and solvent isotope effects for the alpha-chymotrypsin-catalyzed hydrolysis of Suc-Phe-pNA (K(s) = 1 mM, k(2) = 0.04 s(-)(1), and k(3) = 11 s(-)(1)), Suc-Ala-Phe-pNA (K(s) = 4 mM, k(2) = 0.9 s(-)(1), and k(3) = 42 s(-)(1)), and Suc-Ala-Ala-Pro-Phe-pNA (K(s) = 0.1 mM, k(2) = 98 s(-)(1), and k(3) = 71 s(-)(1)). We found that while the van't Hoff plots for K(s) and the Eyring plots for k(3) are linear for all three reactions, the Eyring plots for k(2) are convex, indicating that the process governed by k(2) is complex, possibly involving a coupling between active site chemistry and protein conformational isomerization.

We have found the enrichment in rat liver tritosomes of an enzyme which hydrolyzed the synthetic substrate, Ala-Ala-Phe-pNA, under acidic conditions. The enzyme cleaved the substrate in an exopeptidolytic manner, releasing Ala-Ala-Phe. The enzyme was solubilized from tritosomes and partially purified. Beside Ala-Ala-Phe-pNA, the obtained enzyme was shown to cleave angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) and des-1Tyr-dynorphin A 1-8 (Gly-Gly-Phe-Leu-Arg-Arg-Ile) with tripeptide units. The results obtained suggested that the enzyme preferred an aromatic amino acid at the third position of a substrate peptide.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)-inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2-terminal end of the synthetic peptide Ala-Ala-Phe-p-nitroanilide (pNA) and was not inhibited by inhibitors of metallo-, cysteic-, and aspartic-proteinases, or by those of elastase-, trypsin- and chymotrypsin-like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly-Pro from Gly-Pro-pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N-exo-aminopeptidase activities specifically inhibited by bestatin, was also detected when Ala-, Leu-, Arg- and Lys-pNA were used a substrates.