Fluorescence Study of Chemical Modification of Phosphoenolpyruvate Carboxylase from Crassula argentea

Abstract

The chemical modification of phosphoenolpyruvate carboxylase purified from Crassula argentea leaves was studied using the fluorescence of the extrinsic probe 8-anilino-1-napthalenesulfonate. The effects of ligands on kinefic parameters of phosphoenolpyruvate carboxylase activity, and its response to pH and metal cations, were associated with the binding of the ligands to the enzyme as measured by fluorescence. Binding of the ligands phosphoenolpyruvate, malate, and glucose-6-phosphate revealed by fluorescence measurements corresponds to competitive phenomena observed in kinetic studies. The fluorescence measurements also suggest the involvement of specific amino acids in the binding of a given ligand. Arginyl residues modified by 2,3-butanedione appear to be directly involved in the binding of phosphoenolpyruvate and malate to the active and the inhibition sites, respectively. A histidyl residue was involved in the binding of malate, accounting for the lack of inhibition by malate in kinetic studies of the enzyme treated with diethylpyrocarbonate. Although activity was lost, there was no decrease in the ability of the treated enzyme to bind phosphoenolpyruvate, suggesting that additional histidyl residues are essential for activity although not directly involved in the binding of phosphoenolpyruvate. The lysine reagent trinitrobenzenesulfonate caused a loss of activity and a reduction in malate inhibition and glucose-6-phosphate activation, but these modifications were not related to changes in the ability of the enzyme to bind any of the three ligands. This suggests that lysine residues were not directly involved in the binding of these ligands.

The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize

“…The lower K 0.5 PEP of E229A and the higher K 0.5 PEP of R232Q are consistent with the hypothesis that PEP acts as an allosteric activator. That PEP might bind at the G6P allosteric activation site, in addition to the catalytic site, was first proposed in studies of PEP carboxylase from a CAM plant Rustin and Wedding 1991) involving steady state kinetics and fluorescence experiments. Subsequently, kinetic analysis of the maize enzyme (Rodriguez-Sotres and Munoz-Clares 1990; Tovar-Mendez et al 1998) also led to the suggestion that free PEP (as opposed to the Mg-PEP complex) activates by binding at the G6P allosteric activation site.…”

“…The allosteric properties of the C 4 enzyme include feedback inhibition by malate or aspartate, activation by G6P and, in monocot plants, activation by glycine or alanine. C 4 and CAM PEP carboxylases are activated by a number of phosphorylated metabolites (Rajagopalan et al 1994), including, reportedly, the substrate PEP Rustin et al 1991;Rodriguez-Sotres and Munoz-Clares 1990;TovarMendez et al 1998). While the enzyme serves a prominent photosynthetic role in both C 4 and CAM plants, PEP carboxylase is also known to serve anapleurotic and other non-photosynthetic functions in plants and is also widely distributed in bacteria (Rajagopalan et al 1994).…”