Isotope trapping and kinetic isotope effect studies of rat liver alpha-(2-->6)-sialyltransferase.
A mechanistic study of rat liver alpha-(2-->6) sialyltransferase (ST) is presented that includes isotope trapping experiments and kinetic isotope effects on V/K for the ST-catalyzed reaction of isotopically labeled CMP-N-acetylneuraminate and N-acetyllactosamine. The isotope trapping experiments confirmed that the kinetic mechanism is steady-state random, and further analysis indicated that for this sialyltransferase the experimentally observed isotope trapping ratio (product trapped/substrate released) was equivalent to the commitment to catalysis, Cf, the quantity required to correct the kinetic isotope effects. Cf was found to range from 1.0 (at 1.6 mM LacNAc) to 1.7 (at 100 mM LacNAc). After correction for Cf, the isotope effects were as follows: secondary beta-dideuterium, 1.04-1. 05; anomeric carbon primary 14C, 1.000 +/- 0.004; a small 3H binding effect of 1.016 +/- 0.007 at C9; and a carboxylate carbon secondary 14C isotope effect of 0.998 +/- 0.004. This pattern of KIEs is quite different than observed for solvolysis of CMP-NeuAc [Horenstein, B. A., and Bruner, M. (1996) J. Am. Chem. Soc. 118, 10371-10379]. Based on the results of ab-initio modeling of isotope effects, a hypothesis is presented which reconciles the unusual pattern of KIEs on the basis of binding interactions at the carboxylate carbon.
Bruner
M
M
Department of Chemistry, University of Florida, Gainesville 32611, USA.
Horenstein
B A
BA
D000818:Animals; D002250:Carbon Radioisotopes / Q000378:metabolism; D002384:Catalysis; D003903:Deuterium / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051381:Rats; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D014316:Tritium / Q000378:metabolism; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D002250:Carbon Radioisotopes; D014316:Tritium; D003903:Deuterium; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase