Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

Abstract

Tripeptide derivatives like 3-carboxypropanoylalanyl-alanyMeucine 4-nitroanilide or 3-carboxypropanoylalanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (kcat > 102 s-Z; kcatlKm >-106 s -~ "M-~) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (kcat ~ I s-~-34 s-~). By substituting the N-terminal alanine of the substrates with proline, the catalytic efficiency of the enzymic reaction, by the serine proteases, is diminished by 2-3 orders of magnitude, whereas that by neprilysin and thermolysin decreases only slightly. These effects demonstrate that structural alterations in peptide substrates that impair secondary sub-site interactions with one class of peptidases may enhance the selectivity of the substrates towards another class of peptidases.

A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction

“…Thus, overall, Leu in the P1 0 position is less favorable for LasA protease than elastase. The catalytic coeYcient (0.5 £ 10 3 M ¡1 s ¡1 ) found here for the hydrolysis of SucGly-Gly-Leu-NH-Np by elastase is comparable to that reported for the cleavage of this substrate by thermolysin [24] (1.18 £ 10 3 M ¡1 s ¡1 ). The catalytic coeYcient determined here for the hydrolysis of Suc-Gly-Gly-Phe-NH-Np by elastase (7.6 £ 10 3 M ¡1 s ¡1 ) on the other hand is almost 10-fold higher than that previously determined for thermolysin [24] (0.97 £ 10 3 M ¡1 s ¡1 ).…”

“…Amino-protected tripeptides linked to a chromogenic group at the carboxy terminus, with Phe or Leu at the carboxy terminus serving as the major hydrophobic residue, have been used to determine the activity of thermolysin and other family members (e.g., neprilysin) in two-stage assays whereby the chromophore is released by an externally added aminopeptidase following cleavage of the substrate by the endopeptidase [24,31]. To examine whether this approach may be adapted for assaying LasA protease activity, we Wrst tested whether LasA protease can cleave two such substrates, Suc-GlyGly-Phe-NH-Np and Suc-Gly-Gly-Leu-NH-Np, in which the Gly-Gly sequences correspond to the consensus cleavage site of LasA protease.…”

“…Suc-Gly-Gly-Phe-NH-Np and Suc-Gly-Gly-Leu-NH-Np are cleaved by thermolysin on the carboxyl side of the second Gly residue [24]. P. aeruginosa elastase and thermolysin have essentially the same cleavage speciWcity [2,3].…”

“…Suc-Gly-Gly-Leu-NH-Np was synthesized by a two-step addition of Gly to Leu-NHNp via reaction with the t-butoxycarbonyl amino acid and deblocking of the t-butoxycarbonyl groups with triXuoroacetic acid, followed by succinylation with succinic anhydride [24].…”

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

“…All the substrates presented in Table 1A contain a hydrophobic residue at the P' 1 position (Leu, Phe or Val), an essential condition for NEP activity (1,19). Thus, the excellent kinetic parameters of Abz-DRRLEDDnp and Abz-GGDFLRRV-EDDnp may be also explained by interactions of their groups with subsites on the surface of NEP.…”