Determination of Two Structural Forms of Catalytic Bridging Ligand in Zinc-Phosphotriesterase by Molecular Dynamics Simulation and Quantum Chemical Calculation Chang-Guo Zhan, Osmar Norberto de Souza, Robert Rittenhouse, and Rick L. Ornstein* Contribution from the Pacific Northwest National Laboratory, Battelle-Northwest, EnVironmental Technology DiVision, Mailstop K2-21, Richland, Washington 99352 ReceiVed March 1, 1999 Abstract: Although a three-dimensional X-ray crystal structure of zinc-substituted phosphotriesterase was recently reported, it is uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand is theoretically determined by performing both molecular dynamics simulations and quantum mechanical calculations. All of the results obtained indicate that this critical ligand in the active site of the reported X-ray crystal structure is a hydroxide anion rather than a water molecule and allow us to propose a dynamic “ping-pong” model in which both kinds of structures might exist. Introduction Phosphotriesterase (PTE) from Pseudomonas diminuta cata- lyzes the hydrolysis of organophosphorus pesticides and related nerve agents, i.e., acetylcholinesterase inhibitors, with rate enhancements that approach 10 12 . 1 There is much current interest in understanding how this remarkable enzyme and related binuclear metal complexes catalyze the hydrolysis so ef- fectively. 2 To elucidate the catalytic mechanism, it is first necessary to determine the structure of the active site. Recently, Vanhooke et al. reported a three-dimensional X-ray crystal structure of zinc-substituted PTE complexed with the substrate analogue, diethyl 4-methylbenzylphosphonate. 3 The X-ray crystal structure indicates that the two zinc ions in the active site are separated by 3.3 Å. One of the two bridging ligands for the binuclear metal center is a carbamylated Lys (residue 169). It is uncertain whether the other bridging ligand is a water molecule or a hydroxide ion, since hydrogen atoms cannot be determined by X-ray diffraction techniques. It is expected that this second bridging ligand, hydroxide or water, is directly involved in the catalytic hydrolysis process. Here we theoreti- cally determine the identity of this critical bridging ligand by performing both molecular dynamics (MD) simulations on the solvated PTE-inhibitor complex and quantum chemical cal- culations on simplified models of the active site. On the basis of the theoretical results obtained, a possible dynamic “ping- pong” model is proposed. Calculation Methods MD simulations were carried out on subunit 2 of the PTE dimer, 3 using the SANDER module of AMBER 4.1 4 with the force field * Address correspondence to this author. E-mail: rick.ornstein@pnl.gov. Fax: 509-375-6904. Phone: 509-375-2132. (1) Dumas, D. P.; Caldwell, S. R.; Wild, J. R.; Raushel, F. M. J. Biol. Chem. 1989, 264, 19659. (2) (a) Kuo, J. M.; Chae, M. Y.; Raushel, F. M. Biochemistry 1997, 36, 1982. (b) Hong, S.-B.; Mullins, L. S.; Shim, H.; Raushel, F. M. Biochemistry 1997, 36, 9022. (c) Seo, J. S.; Hynes, R. C.; Williams, D.; Chin, J. J. Am. Chem. Soc. 1998, 120, 9945. (3) Vanhooke, J. L.; Benning, M. M.; Raushel, F. M.; Holden, H. M. Biochemistry 1996, 35, 6020. (4) Pearlman, D. A.; Case, D. A.; Caldwell, J. W.; Ross, W. S.; Cheatham, T. E., III; Ferguson, D. M.; Seibel, G. L.; Singh, U. C.; Weiner, P. K.; Kollman, P. A. AMBER 4.1, University of California, San Francisco, 1995. VOLUME 121, NUMBER 32 AUGUST 18, 1999 © Copyright 1999 by the American Chemical Society 10.1021/ja9906397 CCC: $18.00 © 1999 American Chemical Society Published on Web 07/27/1999