[#1995-03] Characterization of a Thermostable Lysine-Specific Metalloendereptidase from the Fruiting Bodies of a Basidiomycete, Grifola frondosa, Takashi Nonaka et al.

Characterization of a Thermostable Lysine-Specific Metalloendereptidase from the Fruiting Bodies of a Basidiomycete, Grifola frondosa

Takashi Nonaka, Hiroko Ishikawa, Yoichi Tsumuraya, Yohichi Hashimoto, Naoshi Dohmae, and Koji Takio

Department of Biochemistry, Faculty of Science, Saitama University, Saitama, Japan; and Division of Biomolecular Characterization, The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan

A zinc-metalloendopeptidase, MEP, capable of catalyzing specific cleavage of acyl-lysine bonds (-X-Lys-) in polypeptides has been purified 212-fold in a yield of 24.7% from the fruiting bodies of Grifola frondosa, which is a popular edible mushroom called ” maitake ” in Japan. The purified enzyme consists of a single polypeptide chain with an apparent molecular mass of 20 kDa and a pI value of 7.46, contains 1 atom of zinc/molecule and can be inactivated with EDTA or 1,10-phenanthroline. Treatment of MEP with EDTA affords an apoenzyme, whose activity can be fully restored by the addition of Mn2+, Zn2+, Ca2+, or Co2+. Prominent features of MEP are its remarkable heat stability and its high affinity for ₁9- n-glucans and chitin. It hydrolyzes proteins maximally at pH 9-10, liberating only lysyl­peptides. Polylysine and lysine copolymers with alanine, phenylalanine, or glutamic acid can serve as good substrates. Lysylalanine was liberated from bovine insulin and its oxidized B chain by the action of MEP. Mass spectrometric analysis by Frit-FAB MS of the fragments generated from horse heart cytochrome c presented unambiguous evidence to corroborate the specificity of MEP for acyl-lysine bonds.