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.
Kadowaki
H
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Department of Medicine, Boston University School of Medicine, Massachusetts 02118.
Grant
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MA
D000818:Animals; D015703:Antigens, CD; D002240:Carbohydrate Sequence; D002417:Cattle; D005679:G(M3) Ganglioside / Q000096:biosynthesis*; D006028:Glycosphingolipids / Q000378:metabolism*; D007700:Kinetics; D007790:Lactosylceramides; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D012799:Sialyltransferases / Q000378:metabolism*
D015703:Antigens, CD; D005679:G(M3) Ganglioside; D006028:Glycosphingolipids; D007790:Lactosylceramides; C009744:CDw17 antigen; D012799:Sialyltransferases; C021343:haematoside synthetase