A complex array of factors regulate the activity of Arabidopsis thaliana δ1 -pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism

Abstract

The first and committed step in proline synthesis from glutamate is catalyzed by δ¹ -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed K_M values in the micro- to millimolar range, and its activity was inhibited by NADP⁺ , ADP and proline, and by glutamine and arginine at high levels. Mg²⁺ ions were required for activity, which was further stimulated by K⁺ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cations and pyridine dinucleotides.

Keywords: P5C synthetase; amino acid metabolism; cooperative enzyme; feedback and product inhibition; isoenzymes; posttranslational regulation; proline synthesis; pyridine nucleotide pools.