Regulation of Glutamine Synthetase: IX. REACTIVITY OF THE SULFHYDRYL GROUPS OF THE ENZYME FROM ESCHERICHIA COLI

Abstract

Abstract All of the cysteic acid residues (4.6 per subunit) found by amino acid analysis of performic acid-oxidized Escherichia coli glutamine synthetase are present as cysteine in the native enzyme but are buried in the interior of the molecule. They become exposed and accessible to titration by sulfhydryl reagents only when the enzyme is disaggregated into subunits or when divalent cation is removed with ethylenediaminetetraacetate. From the total number of peptides and the number of arginine-containing and 14C-carboxymethylated peptides that are produced upon tryptic digestion of 14C-carboxymethylated glutamine synthetase, it is suggested that the enzyme is composed of identical subunits. However, until an explanation is found for the wide variation in the observed specific radioactivity of the 14C-carboxymethylated peptides thus obtained, this conclusion is still uncertain. Glutamine synthetase can be inactivated by organic mercurials, and the extent of inactivation depends upon the amount of divalent cation in association with the enzyme. Two states of the enzyme are thus defined: relaxed enzyme, without metal, which is susceptible to inactivation and disaggregation by p-chloromercuriphenylsulfonate, and taut enzyme, which has bound manganese and is resistant to such treatment. The metabolic effectors of glutamine synthetase may cause alterations in the rate and extent of inactivation of the enzyme by mercurials. Of the inhibitors that are partially competitive with respect to glutamate, tryptophan diminishes the inactivation by mercurials, cytidine triphosphate enhances this inactivation, and glycine has little effect. Of the compounds that are partially competitive inhibitors with respect to ammonia, histidine is protective against inactivation and glucosamine-6-P has no effect. Similarly, with the noncompetitive inhibitors, AMP decreases the rate of inactivation while carbamyl phosphate and alanine have no influence. These results provide additional support for the occurrence in glutamine synthetase of separate binding sites for each inhibitor, as would be expected from the independent nature of the cumulative feedback inhibition phenomenon.