Biosynthesis of the activated
sulfate donor PAPS (3'-phosphoadenosine 5'-phosphosulfate) involves the sequential action of two
enzyme activities.
ATP sulfurylase catalyzes the formation of APS (adenosine 5'-phosphosulfate) from
ATP and free
sulfate, and APS is then phosphorylated by
APS kinase to produce PAPS. Using rat
chondrosarcoma ATP sulfurylase and
APS kinase, a newly developed assay system, which permits measuring the accumulation of both APS and PAPS in the presence of both
enzyme activities, produces a PAPS/APS ratio corresponding to a "channeling efficiency" of 96%. The velocity of the
APS kinase reaction measured in the overall system with endogenously synthesized APS is 8-fold greater than that of the isolated
kinase reaction using exogenous APS. Most conclusively,
isotope dilution and enrichment experiments show that the APS intermediate does not equilibrate with APS in the bulk medium but remains largely bound in the rat
enzyme system. In contrast, control experiments with a nonchanneled system containing a mixture of the
sulfurylase and
kinase isolated from Penicillium chrysogenum give the results expected for a nonchanneled pathway. These data indicate that APS is channeled between the active sites of
ATP sulfurylase and
APS kinase during the production of PAPS in rat
chondrosarcoma.