Uchida, Y., Tsukada, Y., & Sugimori, T. (1984). Purification and properties of N-acetylneuraminate lyase from Escherichia coli. The Journal of Biochemistry, 96(2), 507-522.
iV-Acetylneuraminate lyase [Ar -acetylneuraminic acid aldolase EC 126.96.36.199] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase.
The enzyme catalyzed the cleavage of TV-acetylneuraminic acid to A-acetylmannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of yV-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60°C. The thermal stability increased up to 75°C in the presence of pyruvate.
No metal ion was required for the enzyme activity, but heavy metal ions such as Ag+ and Hg2+ were potent inhibitors. Oxidizing agents such as ./V bromosuccinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p chloromercuribenzoic acid and mercuric chloride were also potent inhibitors.
The Km values for iV-acetylneuraminic acid and ./V-glycoIylneuraminic acid were 3.6 HIM and 4.3 HIM, respectively. Pyruvate inhibited the cleavage reaction competitively; K\ was calculated to be 1.0 mM. In the condensation reaction, Nacetylglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace iV-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, /3-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate.
The molecular weight of the native enzyme was estimated to be 98,000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6 M guanidine-HCl, the molecular weight was reduced to 33,000, indicating the existence of 3 identical subunits.
The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.