Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

• Published in: FEBS letters 2005-06, Vol.579 (14), p.2991-2994
• Main Authors: Kataoka, Yukiko, Takada, Katsumi, Oyama, Hiroshi, Tsunemi, Masahiko, James, Michael N.G., Oda, Kohei
• Format: Article
• Language: English
• Subjects: 2,4-dinitrophenyl; 2-(N-methylamino)benzoyl; 7-amino-4-methylcoumarin; AMC; benzoyl; Catalysis; Catalytic dyad; D-2,3-diamino propionic acid; Dap; Dnp; Fluorescence resonance energy transfer; fluorescence resonance energy transfer substrate; FRETS; Fungi - enzymology; Fungi - genetics; Glutamic Acid - genetics; Glutamic Acid - metabolism; Glutamic peptidase; Glutamine - genetics; Glutamine - metabolism; Inhibitor; Kinetics; LC–MS; liquid chromatography mass spectrometer; MCA; MeNHBz; Mutation - genetics; Pepstatin-insensitive; Peptide Hydrolases - chemistry; Peptide Hydrolases - classification; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; peptidyl-4-methyl-coumaryl-7-amides; Protease Inhibitors - pharmacology; Scytalidopepsin; Substrate Specificity; Titrimetry
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/j.febslet.2005.04.050

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364–3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P 3 (basic amino acid), P 1 ′ (small a.a.), and P 3 ′ (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. k cat, K m, and k cat/ K m of SGP for d-Dap(MeNHBz)-GFKFF*ALRK(Dnp)- d-R- d-R were 34.8 s −1, 0.065 μM, and 535 μM −1 s −1, respectively. K i of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH 2 for SGP was 1.2 × 10 −10 M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.