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Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.

Original publication




Journal article


Journal of lipid research

Publication Date





953 - 962


EastChem School of Chemistry, University of Edinburgh, Edinburgh EH9 3FJ, United Kingdom; Division of Structural Biology Wellcome Trust Centre for Human Genomics, Oxford OX3 7BN, United Kingdom; Research Complex at Harwell Rutherford Appleton Laboratory, Didcot OX11 0FA, United Kingdom.


Microsomes, Humans, Sphingomonas, Serine, Isotope Labeling, Substrate Specificity, Kinetics, Serine C-Palmitoyltransferase