Abstract
Anthranilate synthase from Euglena gracilis strain Z was purified to electrophoretic homogeneity. The enzyme migrated as a single symmetrical peak on sucrose density gradients with a sedimentation constant of about 5.0 S. The molecular weight determined by gel filtration on Sephadex G-200 was 80,000 +/- 2,500. Polyacrylamide gel electrophoresis of the pure enzyme after sodium dodecyl sulfate denaturation gave a single band at a position corresponding to a molecular weight of 79,000 +/- 2,000. These results suggest that Euglena antranilate synthase is composed of a single polypeptide chain of about 80,000 that possesses both ammonia- and glutamine-dependent activity. All other known glutamine-dependent anthranilate synthases are heterodimers or heterotetramers with the chorismic acid and glutamine binding sites on distinct polypeptide chains.
Highlights
To support the conclusion that the enzyme was totally denatured by the above treatment, samples of the enzyme were either oxidized with performic acid or treated with 6 M guanidine HCl prior to sodium dodecyl sulfate gel electrophoresis [6]
The glutamine-dependent enzyme presumably arose from the NH,utilizing ancestor by aggregation with a molecule that had evolved to catalyze the cleavage of the amide bond of glutamine. Some support for this comes from the observation that ammonia can be utilized for anthranilate synthesis in uiuo [13] and that mutants of E. coli, totally lacking glutamine-dependent activity are capable of growth on minimal medium containing ammonium salts [14]
There is the possibility that polypeptide chain with binding sites for chorismic acid, ammo- the observed fusion is restricted to Euglena
Summary
Polyacrylamide gel electrophoresis of the pure enzyme after sodium dodecyl sulfate denaturation gave a single band at a position corresponding to a molecular weight of 79,000 f 2,000. These results suggest that Euglena anthranilate synthase is composed of a single polypeptide chain of about 80,000 that possesses both ammonia- and glutamine-dependent activity. Component 1 binds chorismate and catalyzes Reaction 2 but cannot by itself utilize glutamine. Anthranilate synthase component 11 is a polypeptide of about 20,000 molecular weight and apparently has no function other than conferring glutamine reactivity upon the anthranilate synthase complex. The structure of the enzyme is different from other anthranilate synthases that have been studied since it appears to be a single polypeptide chain capable of carrying out both Reactions 1 and 2
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