Properties of anthranilate synthetase component II from Pseudomonas putida.
The interaction of Pseudomanas putida anthranilate synthetase Component II (AS II) with glutamine, glutamine analogs, and iodoacetamide has been investigated in order to clarify the initial steps in the mechanism for glutamine utilization. AS II is alkylated and irreversibly inactivated by covalent attachment of approximately 1 eg of L-2-amino-4-oxo-5-chloropentanoic acid (chloroketone) or 1 eq of iodoacetamide. Alkylation of AS II by chloroketone involves initial formation of an enzyme-inhibitor complex having a Ki of 28 muM. Alkylation of AS II by iodoacetamide occurs without initial formation of a reversible complex. In both cases glutamine protects against alkylation and exhibits competitive kinetics. When anthranilate synthetase Component I (AS I) is associated with AS II, the second substrate, chorismate, enhances alkylation of AS II by chloroketone. Alkylation of AS II by iodoacetamide is unaffected by AS I and chorismate. These results suggest a role of chorismate-AS I complex to promote binding of glutamine to AS II or to facilitate conversion of an AS II-glutamine complex to the covalent glutamyl-AS II intermediate. This conclusion is supported by the fact that glutaminase activity of AS II, which requires formation of the glutamyl-AS II intermediate, is stimulated by AS I and chorismate.
D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002827:Chorismic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005973:Glutamine / Q000031:analogs & derivatives / Q000494:pharmacology; D007460:Iodoacetamide / Q000494:pharmacology; D007659:Ketones / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D013045:Species Specificity
D007659:Ketones; D005973:Glutamine; D000878:Anthranilate Synthase; D002827:Chorismic Acid; D008274:Magnesium; D007460:Iodoacetamide