Suc-Ala-Ala-Ala-pNA

The investigation of the catalytic properties of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L., has gained importance, because the enzyme represents a novel, structurally distinct family of metalloproteinases which also includes a human bone morphogenetic protein (BMP1). Astacin releases nitroaniline from succinyl-alanyl-alanyl-alanyl-4-nitroanilide (Suc-Ala-Ala-Ala-pNA), a substrate originally designed for pancreatic elastase. This activity was unexpected since only few metalloproteinases cleave small nitroanilide substrates, and, moreover, the primary specificity of astacin toward protein substrates is determined by short, uncharged amino-acid sidechains in the P'1-position, i.e. the new N-terminus after cleavage. The specificity constants, kcat/Km, for the release of nitroaniline from substrates of the general structure Suc-Alan-pNA (n = 2, 3, 5) and Alan-pNA (n = 1, 2, 3) increase with the number of alanine residues. The longest peptide, Suc-Ala(-)-Ala-Ala-Ala-Ala-pNA, is the only one out of eleven substrates used in this study, which is cleaved at two positions by astacin. The first cleavage yields Suc-Ala(-)-Ala and Ala-Ala-Ala-pNA. From the resulting C-terminal fragment, Ala-Ala-Ala-pNA, a second cut releases nitroaniline. The 1200-fold higher specificity constant observed for the first as compared to the second cleavage in Suc-Ala-Ala-Ala-Ala-Ala-pNA reflects the preference of astacin for true peptide bonds and also the importance of a minimum length of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolyl oligopeptidase (EC 3.4.21.26) has been purified 26,000-fold from bovine lens tissue by anion-exchange chromatography, gel filtration and isoelectric focussing, with an overall yield of 11%. The purified enzyme exhibited an isoelectric point of 4.8, a pH optimum of 7.5 and a molecular mass of 72 kDa under both native and denaturing conditions. The enzyme was inhibited by diisopropylfluorophosphate and N-ethylmaleimide, indicating the presence of an essential serine residue and an -SH group. The purified enzyme hydrolyzes the elastase substrate tboc-ala-ala-pNA an order of magnitude more rapidly than N-suc-gly-pro-MCA. It also hydrolyzes other elastase substrates including N-suc-ala-ala-ala-pNA, N-suc-ala-ala-pNA, N-suc-ala-ala-pro-ala-pNA and tboc-ala-ala-pro-ala-pNA, but at a slower rate. While the purified preparation of the lens enzyme rapidly hydrolyses bradykinin, ile-ser-bradykinin, insulin B chain, angiotensin and val-his-leu-thr-pro-val-glu-lys at the carboxyl side of proline, it does not hydrolyse either lens crysallins or bovine serum albumin. The amino acid sequence (GMFYNAYPQQDG) of a tryptic peptide from the enzyme is identical to the porcine brain prolyl oligopeptidase sequence 184-195. A similar activity has been identified in human lenses using both tboc-ala-ala-pNA and N-suc-gly-pro-MCA as substrates. The molecular weight, substrate specificity, inhibitor susceptibility and amino acid sequence data suggest that the bovine lens prolyl oligopeptidase is similar to prolyl endopeptidase isolated from other sources.

Cucumisin was isolated from prince melon sarcocarp by means of a simple purification procedure. Serine protease inhibitors such as soybean trypsin inhibitor, ovomucoid, and aprotinin had no effect on the enzyme activity. alpha 2-Macroglobulin showed 38% inhibition of the original caseinolytic activity of cucumisin. The favorable synthetic substrates for cucumisin were Glt-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA. The constant (kcat/Km) for Suc-Ala-Pro-Ala-pNA was found to be 30 times greater than that for Suc-Ala-Ala-Ala-pNA. The substrate specificity of cucumisin for oligopeptides and proteins was shown to be broad.