Catalytic strategy of the P—O bond cleaving enzymes EcoRV and myosin Farooq Kiani & Stefan Fischer* Computational Biochemistry, IWR, University of Heidelberg, Germany. Related movies can be downloaded from: http://www.iwr.uni-heidelberg.de/groups/biocomp/fischer * Correspondence to: Stefan.Fischer@iwr.uni-heidelberg.de 1) Abstract Phosphate hydrolysis is an essential reaction in biology. Two enzymes that catalyze this reaction type are EcoRV and myosin: EcoRV is a type II restriction endonuclease from Escherichia coli that protects the host cell from the invading viral DNA by specifically cleaving the DNA at 5'-GAT↓ATC-3' sequence (where ↓ marks the cleavage site). Myosin II is a molecular motor that converts chemical energy derived from the hydrolysis of adenosine triphosphate into mechanical work. Although structure and function of these two enzymes is dissimilar, combined quantum mechanical/classical simulations show that the fundamental catalytic strategy of these two enzymes has striking similarities. Both enzymes cleave their respective substrates through a three-pronged approach (Figure 2): a) P-O bond dissociation by protein stabilization of a trigonal planar metaphosphate, b) water hydrolysis assisted by a surrounding basic protein residue to generate a hydroxyl nucleophile and c) attack of the metaphosphate by hydroxyl group. Energetically most favorable pathways for the hydrolysis of DNA in EcoRV and the hydrolysis of ATP in the molecular motor myosin are discussed in detail. 4) Energetically most favorable pathway for the hydrolysis of phopshoester in EcoRV: Figure 3: Energetically most favorable pathway for the DNA cleavage in EcoRV. R) Reactant state, with the intact phospho-ribose backbone. The hexacoordinate Mg2+ is shown in green dotted lines. The blue arrow indicates the motion of water w2. a0) water w1, activated by the Mg2+, donates a proton to the oxygen of the P-O-C ester bond (blue arrow), thus breaking the P-O bond. d1) Trigonal planar metaphosphate. 2) Mode of action of EcoRV endonuclease and myosin ATPase EcoRV cleaves the P-O-C phosphodiester linkage in the DNA backbone. Once EcoRV recognizes the specific sequence of the invading DNA, a partially-bound closed state is formed. EcoRV progressively binds the DNA tighter (Figure 1), leading to the catalytically competent state in which the enzyme cleaves the poly(ribose-phosphate) backbone of the DNA. Myosin is a molecular motor responsible for muscle contraction and intra-cellular cargo- transport. It uses the hydrolysis of ATP to drive the Lymn-Taylor cycle which describes the cyclic interactions between myosin and the actin filament that produce motion (Figure 1b). It cleaves the P-O-P anhydride linkage in adenosine triphosphate (ATP), which is the common energy currency in biology. Asp90 abstracts a proton from the water w2, thus generating an OH- that can attack the metaphosphate (blue arrows). d2Asp) and d2O2) A proton is shuffled in two steps from Asp90 to water w1. preP) Pre-product state. Numbers in the upper-left corner of each panel give the energy of the structure relative to the reactant (Panel R). Numbers in red give the energy of the transition state between two successive panels. a) b) 5) Energetically most favorable pathway of ATP hydrolysis in myosin: Figure 1a) Mechanism of DNA recognition by the EcoRV endonuclease. b) Structural events during the Lymn-Taylor muscle contraction cycle. 3) Common catalytic strategy in EcoRV and Myosin Figure 4: Energetically most favorable pathway for the hydrolysis of ATP in myosin. R) The reactant state. The thin arrow indicates the breaking of P—O bond. m1) This results in the formation of a metaphosphate (PO3-), m2) in which water Wh re-orients to form a H-bond with Ser181 (thick arrow). g1) Ser181 and the γ-phosphate protons reorient to switch the H-bond network. g2) Helping water Wh rearranges to make the same H-bonds as in the reactant R. P) Product state. The structures corresponding to panels R, m1, m2, g1, g2 and P are the local energy minima. Energies of the structures (relative to the reactant R) are given in parenthesis. Thin arrows indicate electronic rearrangements, thick arrows with a white head indicate motions of the nuclei. 6) Conclusion: Although EcoRV and myosin are two distinct proteins with different substrates, they adopt a strikingly similar catalytic strategy. In both enzymes, i.e. EcoRV and myosin, hydrolysis occurs through a dissociative mechanism in which the initial event in the enzyme is the P-O bond cleavage, prior to nucleophilic attack of the phosphorus atom. This yields a planar metaphosphate group. Both EcoRV and myosin place a general-base to assist the water activation. The attacking water molecule is activated by a proximate general base, which accepts a proton from the water, thereby generating the hydroxyl nucleophile. After generation of both the metaphosphate and the attacking hydroxyl nucleophile in the binding pocket, the eventual hydrolysis in both enzymes is accomplished by the nucleophilic attack of the hydroxyl ion to the planar metaphosphate. Figure 2 Similarity in the catalytic strategies of EcoRV and Myosin. a) Initial P-O bond dissociation and stabilization of a planar trigonal PO3- metaphosphate species. b) General-base assisted generation of a hydroxyl. In EcoRV, the base is the carboxyl group of Asp90, in myosin the base is Glu459. c) Nucleophilic attack of the hydroxyl group onto the phosphorus of the metaphosphate produced in a). References: Kiani, FA, Fischer S. in Molecular Catalysis, Ed. Gade, L. H.; Hofmann, P. 2013, Wiley Publishers. Submitted. Brooks BR, Brucceroli RE, Olafson BD, Swaminathan S, Karplus M. J Comput Chem 1983, 4, 187-217. Imhof P, Fischer S, Smith JC. Biochemistry 2009, 48 (38), 9061–9075. Schwarzl SM, Smith JC, Fischer S. Biochemistry 2006, 45, 5830-5847. Schwarzl SM, Huang D, Smith J, Fischer S. J Comput Chem. 2005, 26, 1359-1371. Fischer S, Karplus M. Chem Phys Lett 1992, 194, 252-261. Imhof P, Noé F, Fischer S, Smith JC. J. Chem. Theory Comput. 2006, 2, 1050 -1056.