Characterization of a thermostable lysine-specific metalloendopeptidase from the fruiting bodies of a basidiomycete, Grifola frondosa.
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 "MAITA-KE" 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 beta-D-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.
Nonaka
T
T
Department of Biochemistry, Saitama University.
Ishikawa
H
H
Tsumuraya
Y
Y
Hashimoto
Y
Y
Dohmae
N
N
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000648:ultrastructure; D002686:Chitin / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D008666:Metalloendopeptidases / Q000187:drug effects / Q000302:isolation & purification*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011509:Proteoglycans / Q000737:chemistry*; D013379:Substrate Specificity
D008670:Metals; D011509:Proteoglycans; D002686:Chitin; D008666:Metalloendopeptidases; D008239:Lysine