Enzymatic 4-O-acetylation of N-acetylneuraminic acid in guinea-pig liver

Sialic acids from the liver and serum of guinea-pig are composed of N-acetylneuraminic acid (Neu5Ac; 85% and 61%, respectively), N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2; 10% and 32%, respectively) and N-glycolylneuraminic acid (Neu5Gc; 5% and 7%, respectively), besides traces of N-glycolyl-4-O-acetylneuraminic acid in serum. The analysis was carried out using thin-layer chromatography, high-performance liquid chromatography, electron impact ionization mass spectrometry, and different enzymes (sialidase, sialate esterase, and sialate-pyruvate lyase after hydrolysis and purification of the sialic acids by ion-exchange chromatography). We showed that this O-acetylation of sialic acids is due to the activity of an acetyl-coenzyme A:sialate-4-O-acetyltransferase (EC 2.3.1.44), which occurs together with sialyltransferase activity in Golgi-enriched membrane fractions of guinea-pig liver. The enzyme operates optimally at 30°C in 70 mM potassium phosphate buffer at pH 6.7 and in the presence of 90 mM KCl with an apparent KM for AcCoA of 0.6 1M and a Vmax of 20 pmol/mg protein x min. The enzyme is inhibited by coenzyme A in a mixed-competitive manner (Ki = 4.2 M), as well as by para-chloromercuribenzoate, MnCl2, saponin and Triton X-100.