β-Cystathionase fromBordetella avium

摘要: Abstract β-Cystathionase (EC 4.4.1.8) from Bordetella avium is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the hydrolysis of L-cystine to yield pyruvic acid, NH3, and thiocysteine. The latter compound highly toxic toward MC3T3-E1 osteogenic cells, rat osteosarcoma other cell lines maintained in tissue culture (Gentry-Weeks, C. R., Keith, J. M., Thompson, J.(1993) Biol. Chem. 268, 7298-7314). Site-directed mutagenesis has established lysine 214 sequence TKYVGGHSD, primarily responsible for internal aldimine binding PLP holoenzyme. Translation DNA β-cystathionase gene (metC) B. avium, reveals 4 cysteine residues/enzyme subunit (Mr = 42,600), spectrophotometric analysis with 4,4′-dithiodipyridine showed there were no disulfide linkages native protein. inhibited by sulfhydryl-reactive agents, including N-ethylmaleimide (NEM). To elucidate mechanism NEM inhibition, each residues at positions 88, 117, 279, 309 was individually replaced alanine or glycine. mutant proteins C88A, C117G, C279G, C309A purified homogeneity, assayed activity, PLP-binding, sensitivity, susceptibility chymotrypsin digestion. activities C88A C279G comparable enzyme, since both forms NEM, neither 88 nor 279 are prerequisite activity. By elimination, 117 must be targets alkylation, resultant inactivation β-cystathionase, -SH reactive agent. Substitution glycine alanine, respectively, yielded inactive C117G C309A. not detectable these proteins, their absorption spectra lacked peak (at 420 nm) characteristic PLP-Schiff base formation. Edman degradation revealed (Mr∼ 36,000) also first 63 amino acids comprising N terminus mutants enhanced Cysteine may reside conformationally sensitive environments, most probably serve structural function. Toxicity assays performed various obtained site-directed only catalytically active cytotoxic cells.