title: beta-Cystathionase from Bordetella avium. Role(s) of lysine 214 and cysteine residues in activity and cytotoxicity.
abstract: beta-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 is highly toxic toward MC3T3-E1 osteogenic cells, rat osteosarcoma cells, and other cell lines maintained in tissue culture (Gentry-Weeks, C. R., Keith, J. M., and Thompson, J. (1993) J. Biol. Chem. 268, 7298-7314). Site-directed mutagenesis has established that lysine 214 of the sequence TKYVGGHSD, is primarily responsible for internal aldimine binding of PLP in the holoenzyme. Translation of the DNA sequence of the beta-cystathionase gene (metC) from B. avium, reveals 4 cysteine residues/enzyme subunit (M(r) = 42,600), and spectrophotometric analysis with 4,4'-dithiodipyridine showed that there were no disulfide linkages in the native protein. beta-Cystathionase is inhibited by sulfhydryl-reactive agents, including N-ethylmaleimide (NEM). To elucidate the mechanism of NEM inhibition, each of the 4 cysteine residues at positions 88, 117, 279, and 309 was individually replaced by alanine or glycine. The mutant proteins C88A, C117G, C279G, and C309A were purified to homogeneity, and each was assayed for enzyme activity, PLP-binding, NEM sensitivity, and susceptibility to chymotrypsin digestion. The activities of mutant proteins C88A and C279G were comparable with that of the native enzyme, and since both forms were inhibited by NEM, neither cysteine 88 nor 279 are prerequisite for enzyme activity. By elimination, cysteine residues 117 and 309 must be the targets for alkylation, and resultant inactivation of beta-cystathionase, by the -SH reactive agent. Substitution of cysteine 117 and 309 with glycine and alanine, respectively, yielded the inactive proteins C117G and C309A. PLP was not detectable in these proteins, and their absorption spectra lacked the peak (at 420 nm) that is characteristic of internal PLP-Schiff base formation. Edman degradation revealed that C117G (M(r) approximately 36,000) also lacked the first 63 amino acids comprising the N terminus of the native protein. The beta-cystathionase mutants C117G and C309A showed enhanced susceptibility to chymotrypsin digestion. Cysteine residues 117 and 309 may reside in conformationally sensitive environments, and in the native enzyme these amino acids most probably serve a structural function. Toxicity assays performed with the various mutant proteins obtained by site-directed mutagenesis established that only catalytically active forms of beta-cystathionase were were cytotoxic for tissue culture cells.
authors.affiliation: Laboratory of Microbial Ecology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: C R
authors.initials: CR
authors.lastname: Gentry-Weeks
authors.forename: J
authors.initials: J
authors.lastname: Spokes
authors.forename: J
authors.initials: J
authors.lastname: Thompson
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001884:Bordetella / Q000201:enzymology*; D002470:Cell Survival / Q000187:drug effects*; D002918:Chymotrypsin; D017931:DNA Primers; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008190:Lyases / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects; D010449:Peptide Mapping; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D012486:Salmonella typhimurium / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D002918:Chymotrypsin; D008190:Lyases; C019965:cystathionine beta-lyase; D005033:Ethylmaleimide