title: Studies on the catalytic mechanism of a glutamic peptidase.
abstract: Scytalidoglutamic peptidase (SGP) is the prototype of fungal glutamic peptidases that are characteristically pepstatin insensitive. These enzymes have a unique catalytic dyad comprised of Gln(53) and Glu(136) that activate a bound water molecule for nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. The hydrolysis by SGP at peptide bonds with proline in the P(1)' position is a rare event among peptidases that we investigated using the series of fluorescence resonance energy transfer peptides, Abz-KLXPSKQ-EDDnp, compared with the series Abz-KLXSSKQ-EDDnp. The preference observed in these two series for Phe and His over Leu, Ile, Val, Arg, and Lys, seems to be related to the structure of the S(1) subsite of SGP. These results and the pH profiles of SGP activity showed that its S(1) subsite can accommodate the benzyl group of Phe at pH 4 as well as the positively charged imidazolium group of His. In the pH range 2 to 7, SGP maintains its structure and activity, but at pH 8 or higher it is irreversibly denatured. The intrinsic fluorescence of the Trp residues of SGP were sensitive to the titration of carboxyl groups having low pK values; this can be attributed to the buried Asp(57) and/or Asp(43) as described in SGP three-dimensional structure. The solvent kinetic isotope effects and the proton inventory experiments support a mechanism for the glutamic peptidase SGP that involves the nucleophilic attack of the general base (Glu(136)) activated water, and establish a fundamental role of the S(1) subsite interactions in promoting catalysis.
authors.affiliation: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-20 São Paulo, Brazil.
authors.forename: Márcia Y
authors.initials: MY
authors.lastname: Kondo
authors.forename: Débora N
authors.initials: DN
authors.lastname: Okamoto
authors.forename: Jorge A N
authors.initials: JA
authors.lastname: Santos
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Kohei
authors.initials: K
authors.lastname: Oda
authors.forename: Bindu
authors.initials: B
authors.lastname: Pillai
authors.forename: Michael N G
authors.initials: MN
authors.lastname: James
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D005658:Fungi / Q000201:enzymology*; D005973:Glutamine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010447:Peptide Hydrolases / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D005973:Glutamine; D001224:Aspartic Acid; D006639:Histidine; D002244:Carbon; D014364:Tryptophan; D011392:Proline; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; C069136:glutamyl endopeptidase II