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.
Keywords: Saccharomyces cerevisiae; dipeptidyl peptidase III; metallopeptidase; molecular dynamics; site-directed mutagenesis.