title: Specificity characterization of the α-mating factor hormone by Kex2 protease.
abstract: Kex2 is a Ca2+-dependent serine protease from S. cerevisiae. Characterization of the substrate specificity of Kex2 is of particular interest because this protease serves as the prototype of a large family of eukaryotic subtilisin-related proprotein-processing proteases that cleave sites consisting of pairs or clusters of basic residues. Our goal was to study the prime region subsite S' of Kex2 because previous studies have only taken into account non-prime sites using AMC substrates but not the specificity of prime sites identified through structural modeling or predicted cleavage sites. Therefore, we used peptides derived from Abz-KR↓EADQ-EDDnp and Abz-YKR↓EADQ-EDDnp based on the pro-α-mating factor sequence. The specificity of Kex2 due to basic residues at P1' is affected by the type of residue in the P3position. Some residues in P1' with large or bulky side chains yielded poor substrate specificity. The kcat/KMvalues for peptides with P2' substitutions containing Tyr in P3were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in kcatand KM, respectively. The pH profile of Kex2 was fit to a double-sigmoidal pH-titration curve. The specificity results suggest that Kex2 might be involved in the processing of the putative cleavage sites in a polypeptide involved in cell elongation, hyphal formation and the processing of a toxin, which result in host cell lysis. In summary, the specificity of Kex2 is dependent on the set of interactions with prime and non-prime subsites, resulting in synergism.
authors.affiliation: Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes - UMC, Mogi das Cruzes, SP, Brazil.
authors.forename: Marcella Araújo
authors.initials: MA
authors.lastname: Manfredi
authors.affiliation: Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes - UMC, Mogi das Cruzes, SP, Brazil.
authors.forename: Alyne Alexandrino
authors.initials: AA
authors.lastname: Antunes
authors.affiliation: Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes - UMC, Mogi das Cruzes, SP, Brazil.
authors.forename: Larissa de Oliveira Passos
authors.initials: LO
authors.lastname: Jesus
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo, SP, 04044-020, Brazil.
authors.forename: Maria Aparecida
authors.initials: MA
authors.lastname: Juliano
authors.affiliation: Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Três de Maio, 100, São Paulo, SP, 04044-020, Brazil.
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.affiliation: Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes - UMC, Mogi das Cruzes, SP, Brazil. Electronic address: wagneras@umc.br.
authors.forename: Wagner Alves de Souza
authors.initials: WA
authors.lastname: Judice
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000072235:Mating Factor / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D043484:Proprotein Convertases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae
keywords: Kex2; Proprotein-processing proteases; Specificity; Synergism; α-mating factor hormone