title: Activity of a peptidase secreted by Phanerochaete chrysosporium depends on lysine to subsite S'1.
abstract: Peptidases are enzymes that catalyze the rupture of peptide bonds. Catalytic specificity studies of these enzymes have illuminated their modes of action and preferred hydrolysis targets. We describe the biochemical characteristics and catalytic specificity of a lysine-dependent peptidase secreted by the basidiomycete fungus Phanerochaete chrysosporium. We attained 5.7-fold purification of a ∼23-kDa neutral peptidase using size-exclusion (Sephadex G-50 resin) and ion-exchange (Source 15S resin) chromatography. Using the Fluorescence Resonance Energy Transfer substrate Abz-KLRSSKQ-EDDnp, we detected maximal activity at pH 7.0 and 45-55°C. The peptidase retained ∼80% of its enzymatic activity for a wide range of conditions (pH 4-9; temperatures up to 50°C for 1h). The peptidase activity was lowered by the ionic surfactants, sodium dodecyl sulfate and cetyltrimethylammonium bromide; the reducing agent, dithiothreitol; the chaotrope, guanidine; copper (II) ion; and the cysteine peptidase-specific inhibitors, iodoacetic acid and N-ethylmaleimide. The peptidase preferred the basic amino acids K and R and high selectivity on S'1subsite, exhibiting a condition of lysine-dependence to catalysis on anchoring of this subsite.
authors: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
authors: Ronivaldo Rodrigues
authors: RR
authors: da Silva
authors: Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
authors: Lilian Caroline Gonçalves
authors: LCG
authors: de Oliveira
authors: Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
authors: Maria Aparecida
authors: MA
authors: Juliano
authors: Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil.
authors: Luiz
authors: L
authors: Juliano
authors: Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors: Jose C
authors: JC
authors: Rosa
authors: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil. Electronic address: hamilton@fcfrp.usp.br.
authors: Hamilton
authors: H
authors: Cabral
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D057056:Cysteine Proteases / Q000737:chemistry* / Q000302:isolation & purification; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D020075:Phanerochaete / Q000201:enzymology; D059748:Proteolysis; D013379:Substrate Specificity
chemical_list: D015853:Cysteine Proteinase Inhibitors; D005656:Fungal Proteins; D057056:Cysteine Proteases; D008239:Lysine
keywords: Cysteine protease; Fungal protease; High specificity