Isotope trapping and kinetic isotope effect studies of rat liver α-(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, C-f, the quantity required to correct the kinetic isotope effects. C-f was found to range from 1.0 (at 1.6 mM LacNAc) to 1.7 (at 100 MM LacNAc). After correction for C-f, the isotope effects were as follows: secondary beta-dideuterium, 1.04-1.05; anomeric carbon primary C-14, 1.000 +/- 0.004; a small H-3 binding effect of 1.016 +/- 0.007 at C9; and a carboxylate carbon secondary C-14 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. Sec. 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.