title: Importance of the three basic residues in the vicinity of the zinc-binding motifs for the activity of the yeast dipeptidyl peptidase III.
abstract: Yeast dipeptidyl peptidase III (yDPP III) is a member of the metallopeptidase family M49 involved in intracellular protein catabolism. Elucidation of the yDPP III crystal structure has pinpointed the zinc-coordinating residues (two His from H(460)ELLGH(465) motif and the second Glu from E(516)ECRAE(521) motif), and several amino acid residues potentially important for catalytic activity whose roles have not been investigated. Here, three putative catalytic residues of the yDPP III, His578, Arg582 and Lys638 were substituted and the resultant single mutants characterized. The replacement of His578 with an asparagine significantly (122-fold) lowered the catalytic efficiency, kcat/Km, for Arg-Arg-2-naphthylamide (Arg2-2NA) hydrolysis, and affinity for hydroxamate inhibitor Tyr-Phe-NHOH (decline by 14-fold). The R582Q mutant exhibited an order of magnitude higher activity with all four dipeptide derivatives examined, compared to the wild type. The molecular dynamics simulations revealed the change in the H-bond networking in the R582Q variant active-site region. The mutation of Lys638, to Leu, slightly increased the specificity constant for Arg2-2NA hydrolysis. However, the affinity for Tyr-Phe-NHOH, and activity for the substrates with uncharged P2 side chains (Ala-Ala-, Ala-Arg- and Phe-Arg-2NA) were dramatically reduced, indicating the importance of the evolutionary conserved salt bridge Lys(638)-Glu(516) for the modulation of DPP III substrate specificity.
authors.affiliation: Division of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia; and Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, 10002 Zagreb, Croatia.
authors.forename: Nina
authors.initials: N
authors.lastname: Jajčanin-Jozić
authors.forename: Sanja
authors.initials: S
authors.lastname: Tomić
authors.forename: Marija
authors.initials: M
authors.lastname: Abramić
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism*
chemical_list: D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D015032:Zinc
keywords: Saccharomyces cerevisiae; dipeptidyl peptidase III; metallopeptidase; molecular dynamics; site-directed mutagenesis