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

Abstract

Phosphoenolpyruvate carboxylases from various organisms contain two conserved lysine residues. In the C4 dicot Flaveria trinervia, one of these residues is Lys 6°°. Converting this Lys 6°° to Arg 6°° or Thr 6°° mainly increased the KI values and but had minimal effect on the Vma X. The Km for PEP, Mg 2+ increased by up to 3-fold in Arg 6°° and Thr 6°° but the Km (HCO]) increased 9-fold in Thr 6°°, suggesting that Lys 6°° might be associated with bicarbonate-binding. This lysine was not obligatory for enzyme activity although the wild-type protein showed higher activity at physiological pH and was less inhibited by malate than the two mutants.Key words: Phosphoenolpyruvate carboxylase; Lysine; Substrate-binding site; Flaveria trinervia binding sites are associated with some of these conserved residues and, since the substrates are anions at neutral pH, it is likely that positively charged residues are involved in the binding sites. Chemical modifications indicate that 2 histidine [6], 2 arginine [7] and 4 lysine [8-10] residues per tetramer are essential for the enzyme function. Modifications of these residues, which may form a substrate (PEP) domain [11], result in enzyme inactivation. In vitro mutagenesis has identified two conserved histidine residues as the PEP-binding sites [12,13]. In this study, we have changed Lys 6°~ in E trinervia, a site previously thought to be involved in PEP-binding [14], to arginine or threonine by techniques of site-directed mutagenesis, and compared their kinetic and regulatory properties with the wildtype enzyme.

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

“…All chemicals, bacterial strains, plasmids, DNA restriction enzymes and modifying enzymes, DNA oligonucleotides, site-directed mutagenesis kit, were as described previously [20].…”

“…The protocol was described previously [20]. The 1.3 kb SalI-BamHI fragment of PEPC cDNA of F. trinervia, which contains the codons for Arg 4~°, Arg r67 and Lys s29, was subcloned into the multicloning site of pALTER-1 for the production of ssDNA.…”

“…For cells carrying mutant cDNAs which encode non-functional PEPC, the minimal medium was supplemented with glutamate (monosodium salt, 1 g/l). The purification of PEPC from transformed PCR1 was described earlier [20]. PEPC activity was determined spectrophotometrically in a coupled system with NADH and malate dehydrogenase at 30°C.…”

“…After 24 h the absorbance increased to 1.54 and 1.22, respectively. It has been observed that E. coli cells containing low PEPC activity take longer to adapt to and grow poorly in minimal medium [20,25]. The decreased cell growth indicated that Gly 829 PEPC might have lower activity than wild-type enzyme.…”

“…Both conserved lysine residues have been mutated to threonine [20] or glycine (present study) by site-directed mutagenesis. These mutations decrease the affinity for PEP, Mg 2+ and bicarbonate, but neither is lethal to enzyme function.…”