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.
Department of Biochemistry and Molecular Biology, Uniformed Services University, Bethesda, MD 20814-4799.
J Lipid Res. 2019 May;60(5):953-962. doi: 10.1194/jlr.M089367. Epub 2019 Feb 21.
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 Our data show that 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 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.
同位素标记经常被用来追踪代谢物在复杂生化途径中的变化，并推断酶催化反应的机制。同位素标记的 l-丝氨酸常用于监测鞘脂生物合成过程中第一个酶，丝氨酸棕榈酰转移酶（SPT）的活性，以及标记下游的细胞代谢物。我们对同位素标记可能对 SPT 催化作用产生的影响感到好奇，因此我们研究了不同的 l-丝氨酸同位素对来自人和细菌的重组 SPT 同工酶的催化活性的影响。我们的数据表明，[2,3,3-D]l-丝氨酸对 SPT 活性显示出明显的同位素效应，而人 SPT 同工酶则没有。这表明，尽管人和 SPT 都催化相同的化学反应，但它们的催化机制可能存在潜在的细微差异。我们的结果表明，可能是人类 SPT 的激活小亚基在这些机制变化中起着关键作用。这项研究还强调，在进行体外和体内研究时，必须考虑底物上同位素标记的类型和位置。
