title: Catalytic stimulation by restrained active-site floppiness--the case of high density lipoprotein-bound serum paraoxonase-1.
abstract: Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168--a key catalytic residue residing >15Å from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.
authors: Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel; Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors: Moshe
authors: M
authors: Ben-David
authors: Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
authors: Joel L
authors: JL
authors: Sussman
authors: Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, S-754 21 Uppsala, Sweden.
authors: Christopher I
authors: CI
authors: Maxwell
authors: Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, S-754 21 Uppsala, Sweden.
authors: Klaudia
authors: K
authors: Szeler
authors: Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, S-754 21 Uppsala, Sweden. Electronic address: kamerlin@icm.uu.se.
authors: Shina C L
authors: SCL
authors: Kamerlin
authors: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address: tawfik@weizmann.ac.il.
authors: Dan S
authors: DS
authors: Tawfik
mesh_terms: D043303:Aryldialkylphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation
chemical_list: D008075:Lipoproteins, HDL; D002265:Carboxylic Ester Hydrolases; C020579:gluconolactonase; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D002118:Calcium
keywords: 192 polymorphism; HDL; PON1; enzyme dynamics; interfacial activation