Studies on the Catalytic Mechanism of a Glutamic Peptidase

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Author Kondo, Marcia Yuri Autor UNIFESP Google Scholar
Okamoto, Debora Noma Autor UNIFESP Google Scholar
Santos, Jorge Alexandre Nogueira Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Oda, Kohei Google Scholar
Pillai, Bindu Google Scholar
James, Michael N. G. Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Gouvea, Iuri Estrada Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Kyoto Inst Technol
Univ Alberta
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.
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Date 2010-07-09
Published in Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 285, n. 28, p. 21437-21445, 2010.
ISSN 0021-9258 (Sherpa/Romeo, impact factor)
Publisher Amer Soc Biochemistry Molecular Biology Inc
Extent 21437-21445
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000279516100030

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