The replacement of Lys620 by serine desensitizes Escherichia coli phosphoenolpyruvate carboxylase to the effects of the feedback inhibitors L-aspartate and L-malate.
Chemical modification of Escherichia coli phosphoenolpyruvate carboxylase (P-pyruvate carboxylase) by 2,4,6-trinitrobenzene sulfonate, a specific reagent for amino groups, causes desensitization to allosteric inhibitors, L-aspartate and L-malate, as well as inactivation. When L-malate is included in the modification mixture, P-pyruvate carboxylase was markedly protected from both desensitization and inactivation [Naide, A., Izui, K., Yoshinaga, T. & Katsuki, H. (1979) J. Biochem. (Tokyo) 85, 423-432]. To determine the lysine residue(s) involved in allosteric inhibition, the lysine residues that were protected from modification by L-malate were investigated by analyzing trinitrophenylated peptides liberated by digestion with glutamyl endopeptidase (V8-protease). The identified residues were Lys491, Lys620, Lys650, and Lys773. Each of these residues was individually replaced with an alanine or serine residue by site-directed mutagenesis to produce mutant enzymes. The mutant enzyme whose lysine residue was replaced with serine ([Ser620]P-pyruvate carboxylase) showed a marked desensitization to L-aspartate and L-malate, while retaining almost the same maximal catalytic activity as the wild-type P-pyruvate carboxylase. Essentially no changes in enzymatic properties were observed for the [Ala491]- and [Ala650]P-pyruvate carboxylases, while for the [Ala620]- and [Ala773]P-pyruvate carboxylases the polypeptides of the expected size were not significantly accumulated in the transformed E. coli cells, presumably due to intracellular degradation.
Yano
M
M
Department of Chemistry, Faculty of Science, Kyoto University, Sakyo-ku, Japan.
Izui
K
K
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005246:Feedback; D007700:Kinetics; D008293:Malates / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D008293:Malates; D001224:Aspartic Acid; C030298:malic acid; D010730:Phosphoenolpyruvate Carboxylase