ATP SULFURYLASE FROM FILAMENTOUS FUNGI - WHICH SULFONUCLEOTIDE IS THETRUE ALLOSTERIC EFFECTOR

Fungal ATP sulfurylase has been reported to be allosterically inhibite d by 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the product of aden osine 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. More over, each sulfurylase subunit contains two sulfonucleotide binding si tes (the catalytic site and a C-terminal, APS kinaselike allosteric si te). Consequently, the possibility that the cooperative effects were c aused 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 conta minating SO42- 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 velo city versus [MgATP] or versus [MoO42-] plots at 100 mu M PAPS were cle arly 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 S2O32-, an inhibitor competitive with MoO42-, activated the reaction at high PAPS and low substrate levels. These re sults are consistent with PAPS serving as a classical allosteric inhib itor. Although APS kinase should be superfluous to the molybdolysis re action, the omission of this enzyme from assay mixtures resulted in ra tes that were higher, the same as, or lower than the corresponding ''p lus APS kinase'' rates, (depending on the fixed level of substrates an d PAPS). Additionally, the ''minus APS kinase'' velocity curves were l ess 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 e nzymes, the cumulative experimental results indicate that PAPS is the true allosteric inhibitor of fungal ATP sulfurylase, binding preferent ially to the T-state allosteric site (or to the allosteric site of the R state inducing the R --> T transition), while APS binds preferentia lly to the R state, probably as a competitive product inhibitor at the catalytic site. If it is assumed that occupancy of the allosteric sit e by any ligand that fits would induce the R --> T transition, then th e results suggest that the allosteric site has evolved to have a highe r affinity for PAPS than for APS (in contrast to real APS kinase). Com puter-assisted simulations allowing for APS and PAPS binding to both t he catalytic and regulatory sites of the hexameric enzyme yielded resu lts that nearly duplicated the experimental curves. (C) 1997 Academic Press, Inc.