Abstract
Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis—the hydrolysis of phosphoserine to serine and inorganic phosphate—in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through a continuous assay that couples phosphate release to the phosphorolysis of a chromogenic nucleoside by the enzyme purine nucleoside phosphorylase (PNP). These assays suffer from numerous drawbacks, and both rely on the detection of phosphate. We describe a new continuous assay that monitors the release of serine by exploiting bacterial serine acetyltransferase (SAT) as a reporter enzyme. SAT acetylates serine, consuming acetyl-CoA and releasing CoA-SH. CoA-SH spontaneously reacts with Ellman’s reagent to produce a chromophore that absorbs light at 412 nm. The catalytic parameters estimated through the SAT-coupled assay are fully consistent with those obtained with the published methods, but the new assay exhibits several advantages. Particularly, it depletes L-serine, thus allowing more prolonged linearity in the kinetics. Moreover, as the SAT-coupled assay does not rely on phosphate detection, it can be used to investigate the inhibitory effect of phosphate on PSP.
Highlights
Phosphoserine phosphatase (PSP; EC 3.1.3.3) is a dimeric enzyme that catalyzes the final and irreversible step of the phosphorylated pathway of L-serine biosynthesis, which contributes most of endogenous L-serine in humans [1]
L-serine is poorly transported across the blood–brain barrier, so that the brain must primarily rely on local L-serine production for protein biosynthesis [2]
The current work was motivated by the quest for a convenient, continuous assay to monitor the PSP reaction based on the detection of L-serine to complement existing detection methods based on the release of inorganic phosphate
Summary
Francesco Marchesani 1 , Erika Zangelmi 2 , Stefano Bruno 1 , Stefano Bettati 3,4 , Alessio Peracchi 2, *. Exploiting Serine Acetyltransferase as the Coupling Enzyme.
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