Protein-peptide interactions are critical for the catalytic function of peptide-modifying enzymes.1 PqqE, a radical S-adenosylmethionine (rSAM) enzyme, plays a central role in the biosynthesis of the peptide-derived cofactor pyrroloquinoline quinone (PQQ) in bacteria.2 It catalyzes the reductive cleavage of SAM to generate a 5′-deoxyadenosyl radical, which initiates a carbon-carbon bond formation on its peptide substrate, PqqA, in the presence of the peptide chaperone, PqqD.3 Despite extensive biochemical studies, the details of the substrate engagement of the enzyme-substrate complex prior to catalysis remain unknown. Here, we present the dynamic interaction model of this quaternary complex using hydrogen-deuterium exchange mass spectrometry (HDX-MS) 4 and nuclear magnetic resonance spectroscopy (NMR). Our results reveal that the binding of individual substrate modulates the conformational dynamics of PqqE, and a pronounced substrate-induced conformational rearrangement only emerges when all four components are assembled. These findings highlight the importance of studying protein-peptide interactions in radical SAM enzymes from a dynamic perspective, offering new insights into how substrate engagement governs catalytic activation in this enzyme class.