Isolation, identification, and synthesis of 2-carboxyarabinitol 1-phosphate, a diurnal regulator of ribulose-bisphosphate carboxylase activity

The diurnal change in activity of ribulose 1,5-bisphosphate (Rbu-1,5-P 2 ) carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing); EC 4.1.1.39] of leaves ofPhaseolus vulgaris is regulated (in part) by mechanisms that control the level of an endogenous inhibitor that binds tightly to the activated (carbamoylated) form of Rbu-1,5-P 2 carboxylase. This inhibitor was extracted from leaves and copurified with the Rbu-1,5-P 2 carboxylase of the leaves. Further purification by ion-exchange chromatography, adsorption to purified Rbu-1,5-P 2 carboxylase, barium precipitation, and HPLC separation yielded a phosphorylated compound that was a strong inhibitor of Rbu-1,5-P 2 carboxylase. The compound was analyzed by GC/MS,13 C NMR, and1 H NMR and shown to be 2-carboxyarabinitol 1-phosphate [(2-C -phosphohydroxymethyl)-D-ribonic acid]. Verification of structure was obtained by comparison of the inhibitory activity of the isolated compound with that of 2-carboxy-D-arabinitol 1-phosphate synthesizedin vitro . This compound (but not 2-carboxy-D-arabinitol 5-phosphate) inhibited Rbu-1,5-P 2 carboxylase in a way that was kinetically identical to that of the isolated, naturally occurring compound. The structure of the isolated compound differs from the Rbu-1,5-P 2 carboxylase transition-state analogue 2-carboxyarabinitol 1,5-bisphosphate only by the lack of the C-5 phosphate group. This difference results in a higher binding constant for the monophosphate (K d = 32 nM) compared with the bisphosphate (K d < 10 pM). The less tightly bound compound acts in a light-dependent, reversible regulation of Rbu-1,5-P 2 carboxylase activityin vivo .