Pathway databases are a means to systematically associate proteins with their functions and link them into networks that describe the reaction space of an organism. Here the Reactome Knowledgebase provides a convenient example to illustrate strategies used to assemble such a reaction space based on manually curated experimental data; approaches to semi-automated extension of these manual annotations to infer annotations for a large fraction of a species’ proteins; and the use of networks of functional annotations to infer pathway relationships among variant proteins that have been associated with disease risk through genome-wide surveys and resequencing studies of tumors.