Anthranilate Synthase from Pseudomonas putida

The anthranilate synthase purified from Pseudomonas putida, is composed of 1 molecule each of two dissimilar protein components. With molecular weights of 63,400 (Component I, or AS I) and 18,000 (Component II, AS II), the pure protein components are free of chorismate mutase, and the enzymes responsible for converting anthranilate to tryptophan but retain trace l-glutaminase activities detectable only by radioisotope assay. Using the determined amino acid composition for each component, the millimolar extinction coefficients for AS I and AS II were calculated to be 5.6 and 0.9, respectively. The experimentally determined millimolar extinction coefficients were 4.4 (AS I) and 1.1 (AS II). AS I and AS II have respective specific activities of 4.7 and 17 in the conversion of chorismate to anthranilate with glutamine as nitrogen source. For this amidetransfer to occur AS II must bind AS I; high glutamine (10 mm) enhances the binding of AS I and II, as does magnesium ion, also required for the amidetransfer. The Km values for chorismate and glutamine in anthranilate synthesis at optimum pH 7.8 are 7 and 600 µm, respectively. At optimum pH 8.8, AS I by itself converts chorismate and ammonia to anthranilate at a rate greater than the maximum for the amidetransfer. The rate of this amination decreases sharply as pH is lowered to neutrality but is unaffected by the addition of AS II. With AS II present and at optimum pH the respective Km values for chorismate and ammonium ion are 0.09 and 26 mm. AS I and II play a joint role in the binding of l-glutamine since [14C]glutamine shows negligible binding to either component alone but does bind to an equimolar mixture of AS I and II with Kd = 14 µm. Additional evidence for this joint role derives from the requirement for the presence of AS I and glutamine for protection of AS II against inactivation by bromopyruvate. Glutamine analogs 6-diazo-5-oxo-l-norleucine and azaserine inactivate amidetransfer but not amination. The inactivation is delayed but not prevented by higher levels of l-glutamine. Inactivation is markedly stimulated by the presence of chorismate. Lineweaver-Burk analysis of the increased inactivation rates showed an apparent Km for chorismate of 10 µm. Although both AS I and AS II must be incubated with 6-diazo-5-oxo-l-norleucine to achieve full inactivation, resolution of 6-diazo-5-oxo-l-norleucine-inactivated anthranilate synthase into its two nonidentical components yields an active AS I and an inactive AS II. The inactivated AS II is reactivated by sulfhydryl compounds such as 2-mercaptoethanol. The importance of an —SH group on AS II placed near the active site of the native complex (AS I + AS II) is suggested.