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The Anthranilate Synthetase-Anthranilate 5-Phosphoribosylpyrophosphate Phosphoribosyltransferase Aggregate

1970; Elsevier BV; Volume: 245; Issue: 6 Linguagem: Inglês

10.1016/s0021-9258(18)63253-3

1083-351X

Ellen J. Henderson, H Zalkin, Lian Hua Hwang,

Folate and B Vitamins Research

Abstract Anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PR transferase) catalyzes the second specific reaction in the tryptophan biosynthetic pathway. This protein is aggregated to anthranilate synthetase Component I, which functions in the catalysis of the preceding reaction in Salmonella tryphimurium. Catalytically active preparations of unaggregated PR transferase had a molecular weight of approximately 87,000. The denatured polypeptide chain had a molecular weight of approximately 63,000, suggesting that the unaggregated enzyme is a single polypeptide chain. Aggregated and unaggregated forms of PR transferase activity were inhibited by tryptophan and required saturating concentrations of substrates for maximal inhibition. Chorismate, a substrate for anthranilate synthetase Component I, antagonized the inhibition of aggregated PR transferase activity by tryptophan but not the tryptophan inhibition of the unaggregated form of the enzyme. Indications of negative cooperativity for anthranilate were observed for aggregated PR transferase in the presence of tryptophan. A partial requirement for Mg2+ was shown for the enzymatic activity and tryptophan inhibition of aggregated PR transferase but not for the unaggregated enzyme. The pH profiles for inhibition by tryptophan differed for the two forms of the enzyme although the pH optima for enzyme activity were similar. The Km values for 5-phosphoribosyl 1-pyrophosphate and anthranilate were 6.7 µm and 8.3 µm, respectively, for the aggregated enzyme and they deviated less than 2-fold for the unaggregated PR transferase. Initial velocity patterns for saturation by substrates of aggregated PR transferase activity appeared parallel but a ping-pong mechanism was excluded by the pattern of PPi product inhibition and lack of exchange of 32PPi into 5-phosphoribosyl 1-pyrophosphate. The patterns of product inhibition by PPi differed for the two forms of PR transferase. It was concluded from presently available evidence that the native anthranilate synthetase-PR transferase aggregate has the composition (anthranilate synthetase)2 (PR transferase)2 and that the PR transferase polypeptide chains, similar to the anthranilate synthetase polypeptide chains, contain sites for substrates and end product inhibitor.