Adenosine Triphosphate Sulfurylase from Penicillium chrysogenum

The molecular weight of Penicillium chrysogenum ATP-sulfurylase (ATP-sulfate adenylyl transferase, EC 2.7.7.4) has been shown to be between 425,000 and 440,000. Other physical parameters determined were: s20,w = 13.0, Stokes' radius = 72 A, D20,w = 2.94 x 10-7 cm3·sec-1, and v = 0.733 cm3·g-1. The carboxymethylated enzyme breaks down to subunits having a molecular weight of approximately 56,000 in the presence of 0.1% sodium dodecyl sulfate. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) reveals that there are eight reactive sulfhydryl equivalents per 440,000 g. This, together with the molecular weight of the subunit and amino acid analysis, suggests that the enzyme is an octamer containing one free sulfhydryl and four disulfides per protomer. Kinetic studies have shown that the actual substrate for the reaction is the ATP-Mg2- complex and that free ATP is a competitive inhibitor with respect to both ATP-Mg2- and MoO42- (Ki = 0.6 to 1.25 mm). The Km for ATP-Mg2- at saturating MoO42- is 4.6 x 10-5 m. The Km for MoO42- at saturating ATP-Mg2- is 1.5 x 10-4 m. In the reverse direction, the Km values for adenosine 5'-phosphosulfate and PPi are 7.1 x 10-6 and 7.7 x 10-5 m, respectively. Initial velocity studies and isotope exchange experiments show that the mechanism of the reaction is of the sequential type. The enzyme is inhibited by adenosine 5'-phosphosulfate (Ki = 4 x 10-5 m) and by sulfide. The inhibition by sulfide is sigmoidal. Sulfide also changes the molybdate concentration dependence from a hyperbolic to a sigmoidal form. The changes in the level of ATP-sulfurylase when mycelia are grown on different sulfur sources suggest that the synthesis of the enzyme is repressed by methionine, or some close metabolite of methionine.