Fungal ATP sulfurylase has been reported to be allosterically inhibited by 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the product of adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the sulfate activation sequence. However, the affinity of ATP sulfurylase for its immediate product, APS, is 1000 times higher than that for PAPS. Moreover, each sulfurylase subunit contains two sulfonucleotide binding sites (the catalytic site and a C-terminal, APS kinase-like allosteric site). Consequently, the possibility that the cooperative effects were caused solely by trace levels of APS, or by APS acting in concert with PAPS could not be dismissed. To identify the true allosteric effector, the molybdolysis reaction kinetics in the absence and in the presence of APS kinase were compared. The rationale was that in the absence of APS kinase, submicromolar levels of APS would be generated from contaminating SO(2-)4 present in the assay components, while in the presence of APS kinase, any APS formed would be converted to PAPS. The results were as follows: In the presence of added APS kinase, the initial velocity versus [MgATP] or versus [MoO(2-)4] plots at 100 microM PAPS were clearly sigmoidal as was the velocity versus [PAPS] plot at subsaturating substrate levels. Hill coefficients were in the range of 2 to 3. Also, low concentrations of S2O(2-)3offn inhibitor competitive with MoO(2-)4, activated the reaction at high PAPS and low substrate levels. These results are consistent with PAPS serving as a classical allosteric inhibitor. Although APS kinase should be superfluous to the molybdolysis reaction, the omission of this enzyme from assay mixtures resulted in rates that were higher, the same as, or lower than the corresponding "plus APS kinase" rates, (depending on the fixed level of substrates and PAPS). Additionally, the "minus APS kinase" velocity curves were less sigmoidal and, in some cases, nearly hyperbolic. The effect of APS kinase was shown to be catalytic in nature. If the data are analyzed in terms of the concerted transition (symmetry) model for allosteric enzymes, the cumulative experimental results indicate that PAPS is the true allosteric inhibitor of fungal ATP sulfurylase, binding preferentially to the T-state allosteric site (or to the allosteric site of the R state inducing the R --> T transition), while APS binds preferentially to the R state, probably as a competitive product inhibitor at the catalytic site. If it is assumed that occupancy of the allosteric site by any ligand that fits would induce the R --> T transition, then the results suggest that the allosteric site has evolved to have a higher affinity for PAPS than for APS (in contrast to real APS kinase). Computer-assisted simulations allowing for APS and PAPS binding to both the catalytic and regulatory sites of the hexameric enzyme yielded results that nearly duplicated the experimental curves.