Purification and characterization of enzyme responsible for N-myristoylation of octapeptide in aqueous solution without ATP and coenzyme a from Pseudomonas aeruginosa

The enzyme that catalyzes N-acyl linkage between myristic acid and the NH2-terminal glycine residue of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-NH2 in aqueous solution without ATP and coenzyme A was found in Pseudomonas aeruginosa. The enzyme was purified from cell-free 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 Kms of the purified enzyme for the substrate myristic acid and the octapeptide were 0.36 and 2.6 mM, 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 Km of myristoyl-CoA was 0.17 mM.