Purification and Characterization of Enzyme Responsible for N-Myristoylation of Octapeptide in Aqueous Solution without ATP and Coenzyme A from Pseudomonas aeruginosa(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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The enzyme that catalyzes N-acyl linkage between myristic acid and the NH_2-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH_2 in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cellfree crude extract using DEAE-Cellulose, Sephadex G-200, CM-Sephadex C-50, and hydroxyapatite column chromatographies, and then purified approximately 1900-fold with about 1.5% recovery of enzyme activity from the crude extract. Finally, the purified enzyme showed a main band on SDS polyacrylamide gel electrophoresis after staining with Coomassie Brilliant Blue. The band corresponded to a molecular mass of approximately 60 kDa. The K_ms of the purified enzyme for the substrate myristic acid and the octapeptide were 0.36 and 2.6mM, respectively. When myristoyl-CoA instead of myristic acid was used as the substrate for the enzyme reaction, myristoyl octapeptide could be synthesized as observed in the case of myristic acid. The K_m of myristoyl-CoA was 0.17mM.
社団法人日本生物工学会の論文
2006-09-25