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Background  Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of  E.coli  GFP-tagged enzymes for cells growing exponentially.    Results  We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of  E.coli’s  metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in  B. subtilis  and found that enzymes localize during exponential growth but not during stationary growth.    Conclusions  We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell’s metabolic network.    Electronic supplementary material  The online version of this article (doi:10.1186/s12918-014-0131-1) contains supplementary material, which is available to authorized users.

Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria