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Biochimie

Title:		Specificity characterization of the alpha-mating factor hormone by Kex2 protease
Author(s):		Manfredi MA; Antunes AA; Jesus LO; Juliano MA; Juliano L; Judice WA;
Address:		"Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes - UMC, Mogi das Cruzes, SP, Brazil. Departamento de Biofisica, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Rua Tres de Maio, 100, Sao Paulo, SP, 04044-020, Brazil. Centro Interdisciplinar de Investigacao Bioquimica, Universidade de Mogi das Cruzes - UMC, Mogi das Cruzes, SP, Brazil. Electronic address: wagneras@umc.br"
Journal Title:		Biochimie
Year:		2016
Volume:		20161005
Issue:
Page Number:		149 - 158
DOI:		10.1016/j.biochi.2016.10.003
ISSN/ISBN:		1638-6183 (Electronic) 0300-9084 (Linking)
Abstract:		"Kex2 is a Ca(2+)-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 downward arrowEADQ-EDDnp and Abz-YKR downward arrowEADQ-EDDnp based on the pro-alpha-mating factor sequence. The specificity of Kex2 due to basic residues at P(1)' is affected by the type of residue in the P(3) position. Some residues in P(1)' with large or bulky side chains yielded poor substrate specificity. The k(cat)/K(M) values for peptides with P(2)' substitutions containing Tyr in P(3) were higher than those obtained for the peptides without Tyr. In fact, P' and P modifications mainly promoted changes in k(cat) and K(M), 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"
Keywords:		"Amino Acid Sequence Amino Acid Substitution Binding Sites/genetics Biocatalysis Catalytic Domain Hydrogen-Ion Concentration Kinetics Mating Factor/genetics/*metabolism Models, Molecular Peptides/genetics/*metabolism Proprotein Convertases/chemistry/geneti;"
Notes:		"MedlineManfredi, Marcella Araujo Antunes, Alyne Alexandrino Jesus, Larissa de Oliveira Passos Juliano, Maria Aparecida Juliano, Luiz Judice, Wagner Alves de Souza eng France 2016/10/11 Biochimie. 2016 Dec; 131:149-158. doi: 10.1016/j.biochi.2016.10.003. Epub 2016 Oct 5"