title: Investigation of a role for Glu-331 and Glu-305 in substrate binding of tripeptidyl-peptidase II.
abstract: 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.
authors.affiliation: Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden.
authors.forename: Ann-Christin
authors.initials: AC
authors.lastname: Lindås
authors.forename: Sandra
authors.initials: S
authors.lastname: Eriksson
authors.forename: Emese
authors.initials: E
authors.lastname: Jozsa
authors.forename: Birgitta
authors.initials: B
authors.lastname: Tomkinson
mesh_terms: D019943:Amino Acid Substitution; D000626:Aminopeptidases; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D009842:Oligopeptides; D018698:Glutamic Acid; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases