Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

Abstract

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.

Keywords: biosynthesis; mechanism; membrane protein; regulation; sphingolipid.

Fig. 1.

A: The proposed mechanism of SPT. Briefly, l-serine binds to the PLP-bound, internal aldimine, displacing the active-site lysine to form the SPT PLP: l-serine external aldimine complex. Binding of palmitoyl-CoA induces a conformational change that causes removal of the α-proton from l-serine to form the PLP: l-serine quinonoid. The quinonoid attacks the palmitoyl-CoA thioester and leads to C-C bond formation, which, after electron rebound, results in CoAS release and formation of the β-keto acid intermediate. Decarboxylation leads to the PLP-bound product quinonoid, which is reprotonated by an active-site acid. The active-site lysine then displaces the 3-KDS product and reforms the internal aldimine with PLP bound. B: Isotope labeling patterns of l-serine substrates used in this study. Each isotopologue is labeled with appropriate heavy atom (deuterium D), ¹³C, or ¹⁵N denoted by an asterisk. l-serine 1 and 5 are referred to as light and heavy, respectively.

Fig. 2.

Rates of SpSPT- and scSPT-catalyzed reactions in the presence of l-serine isotopologues 1–5. A: Initial rate of SpSPT-catalyzed reaction of l-serine isotopologues at varying concentrations with 250 µM palmitoyl-CoA. 1, l-serine; 2, [3,3-D]l-serine; 3, [2,3,3-D]l-serine; 4, [2-¹³C]l-serine; 5, [1,2,3-¹³C, 2-¹⁵N]l-serine. Rate of purified SpSPT (B) and rate of purified scSPT (C). Both B and C were carried out with 10 mM l-serine substrates 1–5 and 250 µM palmitoyl-CoA.

Fig. 3.

Quantification of C17-LCBs produced by yeast membranes. The membranes were prepared from cells expressing scSPT (black bars) or coexpressing LCB1 + LCB2a + ssSPTa (gray bars). Yeast membranes were incubated with l-serine isotopologues 1, 2, and 3, along with 100 μM pentadecanoyl coenzyme-A (C15-CoA) to generate the C17-LCBs. The products were derivatized with AccQ-Fluor reagent and analyzed by HPLC and fluorescent detection.