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Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (>4MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k(cat)(app)/K(M) probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K(M) and k(cat)(app) are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k(cat)(app), possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the mechanism by which tripeptidyl-peptidase II (TPP II) can specifically release tripeptides from the free N-terminus of an oligopeptide. The subtilisin-like N-terminal part of TPP II was modelled using subtilisin as template. Two glutamate residues (Glu-305 and Glu-331) appeared to be positioned so as to interact with the positively charged N-terminus of the substrate. In order to test this potential interaction, both residues were replaced by glutamine and lysine. The catalytic efficiency was reduced 400-fold for the E331Q variant and 20000-fold for the E331K variant, compared with the wild-type (wt). A substantial part of this reduction was due to decreased substrate affinity, since the K(M) for both mutants was at least two orders of magnitude greater than for the wt. This decrease was linked specifically to interaction with the free N-terminal amino group, based on inhibition studies. Glu-305 appears to be essential for enzymatic activity, but the extremely low activity of the E305Q variant prevented an investigation of the involvement of Glu-305 in substrate binding. The present work is, to our knowledge, the first report to investigate a mechanism for a tripeptidyl-peptidase activity through site-directed mutagenesis.

Tripeptidyl-peptidase II (TPP II) is a 138-kDa subtilisin-like serine peptidase forming high molecular mass oligomers of >1000 kDa. The enzyme participates in general protein turnover and apoptotic pathways, and also has specific substrates such as neuropeptides. Here we report the site-directed mutagenesis of amino acids predicted to be involved in catalysis. The amino acids forming the putative catalytic triad (Asp-44, His-264, Ser-449) as well as the conserved Asn-362, potentially stabilizing the transition state, were replaced by alanine and the mutated cDNAs were transfected into human embryonic kidney (HEK) 293 cells. In clones stably expressing the mutant proteins, TPP II activity did not exceed the endogenous activity, thus confirming the essential role of the above amino acids in catalysis. Mutant and wild-type TPP II subunits co-eluted from a gel filtration column, suggesting that the subunits associate and that the native subunit conformation was retained in the mutants. Interestingly, the S449A and a H264A mutant enzyme affected the quaternary structure of the endogenously expressed TPP II, resulting in formation of an active, larger complex of >10,000 kDa.