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

Tripeptide derivatives like 3-carboxypropanoyl-alanyl-alanyl-leucine 4-nitroanilide or 3-carboxypropanoyl-alanyl-alanyl-phenylalanine 4-nitroanilide are very sensitive substrates for neprilysin (k(cat) > 10² s^-1; k(cat)/K(m) ≥ 10⁶ s^-1 · M^-1) and are widely employed in investigations of the enzyme. However, these compounds are also good substrates for the serine proteases chymotrypsin and subtilisin (k(cat) ~1 s^-1-34s^-1). 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.