The enzymes catalyzing the first two reactions in the sulfate activation pathway, ATP-sulfurylase (S) and APS-kinase (K), are fused as ‘KS’ in animals but are fused as ‘SK’ in select bacteria and fungi. We have discovered a novel triple fusion protein of K, S, and pyrophosphatase (P) in several protozoan genomes within the Stramenopile lineage. These triple domain fusion proteins led us to hypothesize that pyrophosphatase enzymes and sulfate activation enzymes physically interact to impact the thermodynamics of the sulfate activation pathway. In support of this hypothesis, we demonstrate through biochemical assays that separately encoded KS and P proteins physically interact and that KS/P complexes activate more sulfate than KS alone. We also conclude on the basis of phylogenetic analyses that all known KS fusions originate from a single fusion event early in the eukaryotic lineage. Strikingly, our analyses support the same conclusion for all known SK fusions. These observations indicate that the patchwork of fused and nonfused S and K genes observed in modern-day eukaryotes and prokaryotes are the result of the two ancestral fusion genes evolving by an assortment of gene fissions, duplications, deletions, and horizontal transfers in different lineages. Our integrative use of genomics, phylogenetics, and biochemistry to characterize pyrophosphatase as a new member of the sulfate activation pathway should be effective at identifying new protein members and connections in other molecular pathways.