Metallopeptidase family M49 is characterized by five conserved sequence regions and the unique motif HEXXGH with two histidines - ligands of the active-site zinc ion. The crystal structure of the yeast ortholog represents a prototype for the whole family. To investigate the role of two invariant amino acid residues, a Glu461 of the zinc-binding motif, and a Tyr327, 21Å from the catalytic zinc center, mutational analysis of the yeast enzyme was performed. The substitution of Glu461 to glutamine decreased kcat for the substrate hydrolysis almost by 10 000-fold. The replacement of Tyr327 by Phe or Ala reduced the catalytic efficiency (kcat/Km) by two orders of magnitude. The affinity for the heptapeptide valorphin was siginificantly lowered in all mutants, in-dicating the contribution of both Glu461 and Tyr327 in substrate binding. Taken together, the effect of mutating Glu461 is consistent with this residue being essential in M49 peptidase catalysis. (doi: 10.5562/cca2107)