The specificity of lysosomal tripeptidyl peptidase-I determined by its action on angiotensin-II analogues

AbstractTripeptidyl peptidase-I (TPP-I) is a lysosomal peptidase which cleaves tripeptides from the N-terminus of peptides. The function of the enzyme is unclear but its importance is demonstrated by the fact that mutations in TPP-I are responsible for late infantile neuronal ceroid lipofuscinosis, a lethal lysosomal storage disease. As a step towards identifying its natural substrates, we have used a series of synthetic peptides, based on angiotensin-II, to explore the effects of peptide chain length and the effects of amino acid substitutions at the P1 and P1′ positions on the rate of catalysis. With the exception of angiotensin-(1–8) (angiotensin-II), which is a relatively poor substrate for TPP-I, the rate of catalysis increases with increasing chain length. Kcat/Km values increase 50-fold between angiotensin-(1–5) and angiotensin-(1–14). TPP-I shows little specificity for the nature of the amino acids in the P1 and P1′ positions, Kcat/Km values varying only 5-fold for a range of substitutions. However, Pro or Lys in the P1 position and Pro in the P1′ positions are incompatible with TPP-I activity. These observations suggest that TPP-I is a non-specific, but essential, peptidase involved in the latter stages of lysosomal protein degradation