succinyl-trialanine-4-nitroanilide and astacin

Astacin, a 23-kDa monomeric metalloprotease from the crayfish digestive tract, and meprin A, a 360-kDa tetrameric metalloprotease from the mouse kidney, are 30% identical in the amino acid sequence of their protease domains (Dumermuth et al., 1991, J. Biol. Chem. 266, 21381). The two were compared kinetically using a variety of substrates and inhibitors. Both enzymes degraded azocasein; meprin A had a twofold higher molar specific activity. Succinyl-Ala-Ala-Ala-p-nitroanilide was cleaved by both enzymes at the Ala-p-nitroanilide bond, indicating that in contrast to many metallopeptidases, peptidases of the astacin family are capable of arylamidolysis. Several peptides from a series of chromogenic and fluorogenic bradykinin analogs, originally designed for mapping the active site of meprin A, were found to be excellent substrates for astacin. Both enzymes cleaved most substrates at the site corresponding to the Phe5-Ser6 bond in native bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). The peptide 2ABz-Arg-Pro-Ile-Phe decreases Ser-Pro-Phe(4-nitro)-Arg was found to have the highest kcat/Km ratio for both peptidases (4.5 x 10(5) M-1 s-1 for meprin A and 2.7 x 10(6) M-1 s-1 for astacin). The two enzymes were found to have several other substrates in common, indicating that some of these peptides could be substrates for the other putative proteases in the astacin family. Acetyl-Arg-Pro-Gly-Tyr-NHOH, an inhibitor which likely binds to the S subsites, was found to act on both enzymes via a predominantly non-competitive mechanism, whereas NHOH-succinyl-Pro-Phe-Arg, which likely binds to the S' subsites, was competitive. Significant specificity differences between astacin and meprin A were seen in substrates and inhibitors with bulky groups in the P1' position. Substrates with Arg, Lys, or Phe in the P1' position were cleaved 10(3)-10(4) times faster by meprin A than by astacin. Actinonin, a naturally occurring peptide hydroxamate with a pentyl group in P1', was a very potent competitive inhibitor of meprin A (Ki = 1.35 x 10(-7) M), but had a 1000-fold weaker affinity for inhibition of astacin. These kinetic differences indicate that the S1' subsite is smaller in astacin than in meprin A and correlate with structural differences seen in the three-dimensional models of the two enzymes.

Topics: Amino Acid Sequence; Animals; Astacoidea; Binding, Competitive; Bradykinin; Caseins; Digestive System; Hydroxamic Acids; Kidney; Metalloendopeptidases; Mice; Models, Chemical; Molecular Sequence Data; Oligopeptides