MECHANISM OF G(M3) GANGLIOSIDE SYNTHESIS - KINETIC-STUDY OF RAT-LIVERCMP-N-NEURAMINATE-LACTOSYLCERAMIDE ALPHA-2,3-SIALYLTRANSFERASE EMPLOYING 19 MOLECULAR-SPECIES OF LACTOSYLCERAMIDE

The apparent K-m and V-max of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase (LacCer alpha 2,3-ST) for lactosylceramid e and CMP-N-acetylneuraminic acid were determined using 19 molecular s pecies of lactosylceramide. The K-m for lactosylceramide varied 6-fold among these molecular species of lactosylceramide, but there was a po or correlation between the K-m for a particular molecular species and the activity of LacCer alpha 2,3-ST for that molecular species. The K- m for CMP-N-acetylneuraminic acid also varied depending on the molecul ar species of lactosylceramide used as substrate, and there was a good correlation between the K-m of LacCer alpha 2,3-ST for CMP-N-acetylne uraminic acid and the activity of the enzyme. Kinetic studies indicate that the reaction mechanism of LacCer alpha 2,3-ST is a sequential, O rdered Bi Bi system. From considerations of the effects of the structu re of the lactosylceramide molecular species on the V-max and K-m for CMP-N-acetylneuraminic acid, it is likely that LacCer alpha 2,3-ST fir st binds lactosylceramide and then CMP-N-acetylneuraminic acid and tha t the rate-limiting step in the reaction is the release of the product G(M3).