The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.
Bibliographical noteFunding Information:
We thank the Israel Structural Proteomics Centre for access to their protein purification and crystallization facilities and Marcin Suskiewicz for his valuable assistance in the kinetic measurements. M.E. is a fellow supported by the Intra-European Fellowship Marie Curie program (grant no. 252836 ). Financial support by the National Institutes of Health ( 2-U54-NS058183 ), the Defense Threat Reduction Agency ( HDTRA1-11-C-0026 ), and the Benoziyo Center for Neuroscience is gratefully acknowledged. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemistry. J.L.S. is the Pickman Professor of Structural Biology. We are very grateful to Reviewer #2 for his or her inspirational comments.
- catalytic promiscuity
- conformational diversity
- enzyme catalysis
- enzyme promiscuity
- structural dynamics