Chemical modification of adenylosuccinate synthetase from Escherichia coli by pyridoxal 5'-phosphate. Identification of an active site lysyl residue.
Incubation of adenylosuccinate synthetase from Escherichia coli with low concentrations of pyridoxal 5'-phosphate (PLP) resulted in a rapid loss of activity (92%), concomitant with the formation of a Schiff base. The inactivation of the enzyme by PLP is apparently first order with respect to PLP. The pseudo-first order rate constant, Kapp, showed a hyperbolic dependence on the concentration of PLP, indicating that a kinetically significant PLP.enzyme intermediate is formed during the inactivation process. Stoichiometry and peptide isolation studies showed that 2 lysine residues were modified during reaction of the enzyme with PLP. The three substrates of adenylosuccinate synthetase (GTP, IMP, and aspartate) showed different effects in their ability to protect the enzyme against PLP inactivation. Complete protection of the enzyme against inactivation can be observed only in the presence of high concentrations of GTP. One lysine residue was protected under these conditions. In contrast to GTP, addition of the other two substrates either alone or together to reaction mixtures did not render protection. Peptide mapping by digesting the enzyme with trypsin revealed that the lysine shielded by GTP is Lys140. Replacing the Lys140 with Ile140 by site-directed mutagenesis resulted in total loss of the activity. These results suggest that Lys140 may play an important role in enzymatic activity.
Dong
Q
Q
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Fromm
H J
HJ
D000265:Adenylosuccinate Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors*; D008239:Lysine; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011732:Pyridoxal Phosphate / Q000494:pharmacology*
D011732:Pyridoxal Phosphate; D008025:Ligases; D000265:Adenylosuccinate Synthase; D008239:Lysine