M. Bruner et Ba. Horenstein, ISOTOPE TRAPPING AND KINETIC ISOTOPE EFFECT STUDIES OF RAT-LIVER ALPHA-(2-]6)-SIALYLTRANSFERASE, Biochemistry, 37(1), 1998, pp. 289-297
A mechanistic study of rat liver alpha-(2-->6) sialyltransferase (ST)
is presented that includes isotope trapping experiments and kinetic is
otope effects on V/K for the ST-catalyzed reaction of isotopically lab
eled CMP-N-acetylneuraminate and N-acetyllactosamine. The isotope trap
ping 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/s
ubstrate 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: second
ary 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 c
arboxylate carbon secondary C-14 isotope effect of 0.998 +/- 0.004. Th
is 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 isoto
pe effects, a hypothesis is presented which reconciles the unusual pat
tern of KIEs on the basis of binding interactions at the carboxylate c
arbon.