RESUMEN
Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O2 and releases H2O2. The oxidation of o-dianisidine by H2O2 catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.
Asunto(s)
Oxidorreductasas de Alcohol , Arabidopsis , Monoéster Fosfórico Hidrolasas , Proteínas Recombinantes , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/química , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Glicolatos/metabolismo , Pruebas de Enzimas/métodos , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Hojas de la Planta/metabolismo , Hojas de la Planta/enzimología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Espectrofotometría/métodosRESUMEN
Phosphoglycerate mutases (PGAMs) catalyse the reversible isomerisation of 3-phosphoglycerate and 2-phosphoglycerate, a step of glycolysis. PGAMs can be sub-divided into 2,3-bisphosphoglycerate-dependent (dPGAM) and -independent (iPGAM) enzymes. In plants, phosphoglycerate isomerisation is carried out by cytosolic iPGAM. Despite its crucial role in catabolism, little is known about post-translational modifications of plant iPGAM. In Arabidopsis thaliana, phosphoproteomics analyses have previously identified an iPGAM phosphopeptide where serine 82 is phosphorylated. Here, we show that this phosphopeptide is less abundant in dark-adapted compared to illuminated Arabidopsis leaves. In silico comparison of iPGAM protein sequences and 3D structural modelling of AtiPGAM2 based on non-plant iPGAM enzymes suggest a role for phosphorylated serine in the catalytic reaction mechanism. This is confirmed by the activity (or the lack thereof) of mutated recombinant Arabidopsis iPGAM2 forms, affected in different steps of the reaction mechanism. We thus propose that the occurrence of the S82-phosphopeptide reflects iPGAM2 steady-state catalysis. Based on this assumption, the metabolic consequences of a higher iPGAM activity in illuminated versus darkened leaves are discussed.