Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase

Abstract

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the e-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. ZI, 153.5-1.558 (1962)l provides clear evidence that at pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s).One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isot hiocyanate.In the doubly labeled protein non-radiative singlet-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+_ 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8 -3 .l, 1.9 -2.1, 2.9 -3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8 -5.1 nm from the donor site and the third site being 6.4 nm apart from the donor.Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed.Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.Photosynthetic C 0 2 fixation by C4 plants involves a specialized metabolic pathway, the first step of which is the irreversible formation of oxaloacetate from phosphoenolpyruvate (P-pyruvate) and bicarbonate, catalyzed by Ppyruvate carboxylase [l -31. This enzyme, localized in the cytoplasm of leaf mesophyll cells [4], has been described as an oligomeric protein composed of four identical subunits of M , 100000 [5].Cysteine [6-81, histidine [9], arginine [lo] and lysine [ l l , 121 residues have been reported to be essential for catalytic activity.It has been shown that several metabolites and leaf illumination are responsible for the regulation of the carboxylase activity [13 -171. The extent of light activation and kinetic properties such as affinity constants for substrates and effectors are markedly affected by pH [9, 10, 18 -201. Correspondence to C. S. Andreo, Centro de Estudios Fotosintkticos y Bioquimicos, Suipacha 531, RA-2000 Rosario, ArgentinaAbbreviations. P-pyruvate, phosphoenolpyruvate ; Dns-...

Site‐directed mutagenesis of Flaveria trinervia phosphoenolpyruvate carboxylase: Arg450 and Arg767 are essential for catalytic activity and Lys829 affects substrate binding

“…tions suggest that two lysines per subunit are accessible for chemical modifications and one of these is essential for the catalytic activity [15][16][17]. We previously reported that one of the conserved lysines, Lys 6°° in F trinervia, is likely to be linked to bicarbonate binding [20], a finding at variance with chemical modification studies [19].…”

“…However, it is possible that the loss of activity may by the result of several residues being simultaneously modified by pyridoxal 5'-phosphate [19]. Furthermore, the reagents used to modify lysine residue of PEPC, i.e., pyridoxal 5'-phosphate [15], eosin isothiocyanate [16] and dansyl chloride [17], are large cyclic molecules which could sterically impede the entry of substrate to the active site. In contrast, substitution of lysine by threonine or glycine is non-obstructive and allows the roles of the residues to be assessed directly.…”

“…Selective modifications of lysine residues by pyridoxal 5'-phosphate [15], eosin isothiocyanate [16], dansyl chloride [17] and fluorescein isothiocyanate [18] suggest that one or two lysine residues per subunit can be *Corresponding author. Fax: (61) (8) modified and that one of these is essential for the activity [15].…”

Site‐directed mutagenesis of Lys600 in phosphoenolpyruvate carboxylase of Flaveria trinervia: its roles in catalytic and regulatory functions

“…Chemical modifications of amino acid residues have shown that some histidine, lysine and arginine residues are essential for the catalytic activity of PEPC [6][7][8][9][10]. Although their precise locations can not be determined by this approach, the conserved residues are likely to be associated with substrate-binding sites and they have served as a guide for site-directed mutagenesis to identify the residues involved.…”

Maize Leaf Phosphoenolpyruvate Carboxylase

“…With respect to the molecular structure of the C4 plant enzyme, only recently some reports have presented evidence on the possible spatial arrangement of the carboxylase molecule (1,25,27). Further evidence comes from the dissociation observed after chemical modification of histidine or thiol groups (28) or after exposure to increasing ionic strength (26).…”