Purification and Characterization of Streptomyces griseus Catechol O-Methyltransferase
AUTOR(ES)
Dhar, Kajari
FONTE
American Society for Microbiology
RESUMO
A soluble (100,000 × g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-l-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7.5 and 35°C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the Km for 6,7-dihydroxycoumarin was 500 ± 21.5 μM, and that for S-adenosyl-l-methionine was 600 ± 32.5 μM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-l-methionine, with a Ki of 224 ± 20.6 μM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg2+, p-chloromercuribenzoic acid, and N-ethylmaleimide.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=92394Documentos Relacionados
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