Ligand binding can change the pKa of protein residues and influence enzyme catalysis. Herein, we report three sub-Angstrom resolution X-ray crystal structures of CTX-M β-lactamase, representing three stages of the enzymatic pathway, apo protein (0.79 Å), pre-covalent complex (0.89 Å), and acylation transition state analog (0.84 Å). The binding of a non-covalent ligand induces a proton transfer from the catalytic Ser70 to the general base Glu166, and the formation of a low-barrier hydrogen bond (LBHB) between Ser70 and Lys73. QM/MM reaction path calculations determined the proton transfer barrier between Ser70 and Lys73 to be 1.53 kcal/mol, further confirming the presence of a LBHB. This LBHB is absent in the other two structures. Our data represents the first evidence of a direct and transient LBHB stabilizing a nucleophilic serine, as hypothesized by Cleland and Kreevoy. These results have important implications for the study of enzyme mechanisms as well as protein-inhibitor interactions.

Ligand-Induced Proton Transfer and Low-Barrier Hydrogen Bond Revealed by X-ray Crystallography / Chen, Yunichols; Hargis, Jacqueline C.; Sanishvili, Ruslan; Jaishankar, Priyadarshini; Defrees, Kyle; Smith, Emmanuel W.; Wang, Kenneth K.; Prati, Fabio; Renslo, Adam R.; Woodcock, H. Lee; Chen, Yu. - In: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. - ISSN 0002-7863. - STAMPA. - 137:25(2015), pp. 8086-8095.

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