Phosphoenolpyruvate carboxylase of Escherichia coli. The role of lysyl residues in the catalytic and regulatory functions.
Phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of E. coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased. The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP.
D001224:Aspartic Acid / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007773:Lactates / Q000494:pharmacology; D008239:Lysine; D008274:Magnesium / Q000494:pharmacology; D008293:Malates / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*; D013053:Spectrophotometry; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology
D007773:Lactates; D008293:Malates; D001224:Aspartic Acid; D014302:Trinitrobenzenesulfonic Acid; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium; D008239:Lysine