Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions. Conformational heterogeneity of arginine side chains was quantified for states along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK. The catalytically essential groups were found to be remarkably rigid in a transition state analogue complex, indicating that the enzyme evolved to restrict the conformational freedom along its reaction path, which allows the phosphoryl transfer to occur selectively by avoiding side reactions.