Is metabolism spatially optimized? Structural modeling of consecutive enzyme pairs reveals no evidence for spatial optimization of catalytic site proximity

Abstract Metabolic pathways are often hypothesized to benefit from the spatial organization of enzymes, facilitating substrate transfer through mechanisms such as metabolic channeling or metabolon formation. However, it remains unclear whether the spatial proximity of catalytic sites represents a general organizational principle of metabolism or is restricted to specific pathways. Here, we investigate whether consecutive enzymes in metabolic pathways, when physically interacting, exhibit structurally optimized arrangements that minimize distances between their catalytic sites, thereby increasing metabolite transfer efficiency from one enzyme to the next. We first evaluated the ability of current protein-protein interaction prediction methods, including AlphaFold2, AlphaFold3, ESMFold, and HDOCK, to model weak and transient interactions using a benchmark dataset of 112 low-affinity protein dimers from PDBbind. AlphaFold-based approaches performed best in recovering correct interaction geometries, while ESMFold showed limited performance. We further assessed several confidence metrics and identified ipTM, ipSAE, and VoroIF-GNN as the most informative predictors of correct interaction conformations. In addition to simple Euclidean distance metrics, we developed a computational procedure to estimate shortest accessible space paths between catalytic sites in predicted enzyme-enzyme complexes. Applying this framework to 107 consecutive enzyme pairs in E.coli revealed an increased tendency for consecutive enzymes to interact, but no systematic evidence that interacting enzymes position their catalytic sites in spatially optimized configurations. In the predicted complex conformations, catalytic sites tend not to be positioned closer than expected at random. The developed computational workflow provides a general framework for analyzing structural aspects of metabolic organization.