Mechanism of GM3 ganglioside synthesis. Kinetic study of rat liver CMP-N-neuraminate:lactosylceramide alpha 2,3-sialyltransferase employing 19 molecular species of lactosylceramide.

The apparent Km and Vmax of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase (LacCer-alpha 2,3-ST) for lactosylceramide and CMP-N-acetylneuraminic acid were determined using 19 molecular species of lactosylceramide. The Km for lactosylceramide varied 6-fold among these molecular species of lactosylceramide, but there was a poor correlation between the Km for a particular molecular species and the activity of Lac-Cer alpha 2,3-ST for that molecular species. The Km for CMP-N-acetylneuraminic acid also varied depending on the molecular species of lactosylceramide used as substrate, and there was a good correlation between the Km of Lac-Cer alpha 2,3-ST for CMP-N-acetylneuraminic acid and the activity of the enzyme. Kinetic studies indicate that the reaction mechanism of LacCer alpha 2,3-ST is a sequential, Ordered Bi Bi system. From considerations of the effects of the structure of the lactosylceramide molecular species on the Vmax and Km for CMP-N-acetylneuraminic acid, it is likely that LacCer alpha 2,3-ST first binds lactosylceramide and then CMP-N-acetylneuraminic acid and that the rate-limiting step in the reaction is the release of the product GM3.