The enzyme OmpT of the outer membrane of Escherichia coli shows proteolytic activity and cleaves peptides and proteins. Using molecular dynamics simulations in a fully hydrated lipid bilayer on a time scale of hundreds of nanoseconds, we draw a detailed atomic picture of substrate recognition in the OmpT-holo enzyme complex. Hydrogen bonds and salt bridges are essential for maintaining the integrity of the active site and play a central role for OmpT in recognizing its substrate. Electrostatic interactions are critical at all stages from approaching the substrate to docking at the active site. Computational alanine scanning based on the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach confirms the importance of multiple residues in the active site that form salt bridges. The substrate fluctuates along the axis of the β-barrel, which is associated with oscillations of the binding cleft formed by the residue pairs D210-H212 and D83-D85. Principal component analysis suggests that substrate and protein movements are correlated. We observe the transient presence of putative catalytic water molecules near the active site, which may be involved in the nucleophilic attack on the cleavable peptide bond of the substrate.