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Enzyme Kinetic Characterization and Substrate Specificity of Schizophyllum commune Metacaspases
Author(s) -
Nguyen Le Minh,
Fox Kristin M.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.781.12
Subject(s) - cleave , biochemistry , amino acid , arginine , peptide , proteolysis , enzyme , chemistry , peptide sequence , lysine , cysteine , deubiquitinating enzyme , biology , ubiquitin , gene
Metacaspases are heterodimeric enzymes that belong to the family of cysteine‐dependent proteases, making them distantly related to metazoan caspases and potentially involved in programmed cell death in fungi, protozoa, and plants. Although metacaspases share structural characteristics with caspases, they lack aspartate specificity and instead show a preference for cleaving peptide bonds after arginine or lysine residues at P 1 . In our research, the five Type 1 Schizophyllum commune metacaspases 1–5 ( Sc Mc1‐5) exhibit arginine and lysine specificity. However, the question of whether the enzymes show preference towards any specific amino acids that come before the cleavage site remains unanswered. To investigate the importance of substrate amino acid sequence for substrate specificity, we performed in‐vitro fluorescent activity assays comparing how Sc MC1‐5 cleave various synthetic peptide substrates. Specifically, we examined the effect of amino acid size, charge and polarity on peptide cleavage. The results indicate that Sc MC1‐5 demonstrate a preference to cleave more readily after arginine than lysine. They also exhibit significantly higher hydrolytic activity before small residues like glycine relative to larger residues like phenylalanine. Studying such differences in substrate specificity can be relevant for determining the types of proteins metacaspases cleave in vivo . In addition, we conducted enzymatic characterization to analyze Sc MC1‐5 kinetics. Sc MC1 showed a significantly larger K m , but the V max values for all the S. commune metacaspases were similar. Examining the differences among the metacaspases can further our understanding of their individual, physiological functions. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .