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The Anthranilate Synthetase-Anthranilate-5-Phosphoribosylpyrophosphate Phosphoribosyltransferase Aggregate

1970; Elsevier BV; Volume: 245; Issue: 15 Linguagem: Inglês

10.1016/s0021-9258(18)62924-2

1083-351X

Hiroshi Nagano, H Zalkin, Ellen J. Henderson,

Enzyme Structure and Function

Abstract The reaction of the glutamine analogue, 6-diazo-5-oxo-l-norleucine (DON), with the bifunctional, regulatory enzyme aggregate of the tryptophan pathway, anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase), and with the anthranilate synthetase Component I protein of the aggregate was studied. DON inhibits anthranilate synthetase Component I activity and glutamine- and NH3-dependent anthranilate synthetase activities of anthranilate synthetase-PR transferase. DON inactivates glutamine-dependent anthranilate synthetase activity of the enzyme aggregate, but has less effect on the NH3-dependent anthranilate synthetase activity of the aggregate and does not inactivate anthranilate synthetase Component I. Inactivation of anthranilate synthetase activity of anthranilate synthetase-PR transferase by DON is dependent upon the substrate chorismate, thus providing evidence for ordered binding of first chorismate and then DON (or glutamine). Inactivation of anthranilate synthetase by DON is prevented by the feedback inhibitor, tryptophan. It is suggested that tryptophan evokes feedback inhibition, at least in part, by either preventing chorismate from binding or by preventing the conformational change needed to allow glutamine to bind. The two components of the aggregate, anthranilate synthetase Component I and PR transferase, can be separated by disc gel electrophoresis in 8 m urea. Following treatment of anthranilate synthetase-PR transferase with 14C-DON, most of the radioactivity is incorporated into PR transferase. It therefore appears that the two substrates for anthranilate synthetase bind to different polypeptide chains, chorismate to anthranilate synthetase Component I and glutamine to PR transferase. Approximately 2 moles of 14C-DON bind per mole of enzyme; this suggests a subunit composition of (anthranilate synthetase Component I)2 (PR transferase)2. Titrations with 5,5'-dithiobis(2-nitrobenzoic acid) indicate that DON alkylates 2 cysteine residues per enzyme molecule. A glutaminase activity has been detected in purified preparations of anthranilate synthetase-PR transferase. Glutaminase activity is stimulated by chorismate and inhibited by tryptophan. It is suggested that this activity serves to transfer the amide of glutamine from the site on PR transferase to the NH3 site on anthranilate synthetase Component I. A reaction mechanism which accommodates the experimental data is proposed.