RESUMO
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.
Assuntos
Aminopeptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Proteínas de Drosophila/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminopeptidases/biossíntese , Aminopeptidases/genética , Animais , Domínio Catalítico , Sequência Conservada , Dipeptidil Peptidases e Tripeptidil Peptidases/biossíntese , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Drosophila/enzimologia , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Escherichia coli , Humanos , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Ligação Proteica , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Homologia Estrutural de Proteína , Especificidade por Substrato , Subtilisinas/químicaRESUMO
Tripeptidyl-peptidase II (TPP II) is a giant cytosolic peptidase with a proposed role in cellular protein degradation and protection against apoptosis. Beside its well-characterised exopeptidase activity, TPP II also has an endopeptidase activity. Little is known about this activity, and since it could be important for the physiological role of TPP II, we have investigated it in more detail. Two peptides, Nef(69-87) and LL37, were incubated with wild-type murine TPP II and variants thereof as well as TPP II from human and Drosophila melanogaster. Two intrinsically disordered proteins were also included in the study. We conclude that the endopeptidase activity is more promiscuous than previously reported. It is also clear that TPP II can attack longer disordered peptides up to 75 amino acid residues. Using a novel FRET substrate, the catalytic efficiency of the endopeptidase activity could be determined to be 5 orders of magnitude lower than for the exopeptidase activity.