Comparison of formation of reactive conformers for ؊ the S 2 displacements by CH CO in water and by N ؊ 3 2 Asp124-CO2 in a haloalkane dehalogenase Sun Hur, Kalju Kahn, and Thomas C. Bruice* Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106 Contributed by Thomas C. Bruice, November 26, 2002 ؊ The SN2 displacement of Cl from 1,2-dichloroethane by acetate ؊ (CH3CO2 ) in water and by the carboxylate of the active site aspartate in the haloalkane dehalogenase of Xanthobacter auto- thropicus have been compared by using molecular dynamics sim- ulations. In aqueous solution, six families of contact-pair structures (I–VI) were identified, and their relative concentrations and disso- ciation rate constants were determined. The near attack conform- ers (NACs) required for the SN2 displacement reaction are mem- ؊ bers of the IV (CH3COO ⅐⅐⅐CH2(Cl)CH2Cl) family and are formed in the sequence II3III3IV3NAC. The NAC subclass is defined Scheme 1. by the OCOO؊⅐⅐⅐COCl contact distance of <3.41 Å and the OCOO؊⅐⅐⅐COCl angle of 157–180°. The mole percentage of NACs and adopted basis Newton-Raphson (ABNR) methods (7). An is 0.16%, based on the 1 M standard state. This result may be MD simulation was performed on the energy-minimized system compared with 13.4 mole percentage of NACs in the Michaelis with the position of carboxyl carbon of the acetate fixed at the complex in the enzyme. It follows that NAC formation in the BIOCHEMISTRY center of the box. A periodic boundary condition was used to enzyme is favored by 2.6 kcal͞mol. Because reaction coordinates simulate a continuous water pool. The SHAKE algorithm was used from S to TS, both in water and in the enzyme, pass via NAC (i.e., to constrain bonds containing hydrogens to their equilibrium S 3 NAC 3 TS), the reduction in the S 3 NAC barrier by 2.6 kcal͞ lengths (8). The Verlet leapfrog algorithm was used to integrate mol accounts for Ϸ25% of the reduction of total barrier in the the equations of motion (9). A time step of 1.5 fs was used, and S 3 TS (10.7 kcal͞mol). The remaining 75% of the advantage of the the nonbonded list was updated every 20 time steps. The enzymatic reaction revolves around the efficiency of NAC 3 TS nonbonded interactions were cut off at 10 Å. The Coulombic step. This process, based on previous studies, is discussed briefly. term was cut off by using a force shifting function, and the Lennard-Jones term was cut off with a switching function. The anthobacter autothropicus haloalkane dehalogenase (DhlA) system was initially coupled to a 200 K heat bath for 15 ps by Ϫ Xcatalyzes the SN2 displacement of the halogen substituent using a coupling constant of 5 s 1. The system was subsequently Ϫ from haloalkanes by Asp-CO . This reaction can be compared 2 Ϫ coupled to a heat bath at 300 K for the rest of the simulation Ϫ Ϫ with the reaction in water with CH3CO2 (AcO ) as a nucleo- (23 ns) by using a coupling constant of 5 s 1. The pressure was ⌬ phile (Scheme 1). The activation barrier ( G ) for the displace- constantly maintained by a Berendsen algorithm (10) using an Ϫ ͞ Ϫ Ϫ ment of Cl from dichloroethane (DCE) is 15.3 kcal mol for the isothermal compressibility of 4.63 ϫ 10 5 atm 1 and a pressure Ϸ ͞ enzymatic reaction (1) and 26 kcal mol for the nonenzymatic coupling constant of 5.0 ps. Coordinates were saved every 100 counterpart in water (2). The first goal of this study is to devise time steps. means of determining the time-dependent mechanism of form- When analyzing the trajectory, a configuration of AcOϪ and ing contact pairs from two separate reactants in water that satisfy DCE is classified as a contact pair when the closest intermolec- the structural restraints of a near attack conformation (NAC). ular distance is within the van der Waals distance between two The second objective is to evaluate the contribution of NAC interacting atoms. The van der Waals radii used are 1.92 Å for formation to the kinetic advantage of the DhlA reaction over the sp3 carbon, 1.76 Å for sp2 carbon in acetate, 1.49 Å for acetate model reaction in water. These procedures can be generally oxygen, and 1.75 Å for chlorine (11). applied to a study of various enzymatic catalysis over a water reaction. Results Ϫ Methods Interactions between AcO and DCE (0.214 M for each) were investigated in water by using 23 ns MD simulation. The follow- Molecular Dynamics (MD) Simulation of 1,2-DCE and Acetate in Water. ing results are based on the analysis of structures at every 0.15 To compare ground state structures of reactants in water with ps of this MD trajectory. Reported numerical values in this paper those in the enzyme active site, an MD simulation was per- are based on the correction toa1Mstandard state of both formed on the system consisting of 1,2-DCE and an acetate ion Ϫ reactants. (AcO ) in a box of TIP3P water (3). Standard CHARMM (version Ϫ force field was used for AcO although OPLS force field Structures of Contact Pairs (AcO؊⅐DCE) in Water. Ina1Maqueous (27 parameters were adopted for DCE. The torsional parameters for solution of AcOϪ and 1,2-DCE, collision dimers are present 43% DCE were adjusted so that only one of the two gauche confor- of the time. Once these dimers form, the partners remain mations was sampled. It is known that for the SN2 displacement together for Ϸ25 ps on average and in few cases up to Ϸ80 ps. of ClϪ, DCE should be in a gauche conformation (4–6). After the reactants were placed in a water box of size 20 ϫ 20 ϫ 20 Å3, any TIP3P water molecule whose oxygen was within 2.6 Å from any Abbreviations: DhlA, Xanthobacter autothropicus haloalkane dehalogenase; DCE, dichlo- atom of the reactants was deleted, and the system was energy- roethane; AcOϪ, acetate; NAC, near attack conformer; MD, molecular dynamics. minimized by using a combination of the steepest descent (SD) *To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.242721799 PNAS ͉ March 4, 2003 ͉ vol. 100 ͉ no. 5 ͉ 2215–2219 Downloaded by guest on September 27, 2021 Ϫ O Ϫ⅐⅐⅐ O Fig. 1. Six contact pairs of ClCH2CH2Cl and CH3CO2 . NAC is subspecies of IV where the angle of approach COO C Cl is within the range of 157–180°. Atoms are colored by using standard atom representation: O (red), C (gray), H (white), Cl (green). Ϸ During this 25- to 80-ps time, the type of contact changes every for its dissociation (kdiss). The kdiss for contact pair decomposi- few picoseconds. Fig. 1 shows structures of the various dimers. tion was obtained from the statistics of the length of time for The formation of six contact pairs can be observed to occur at each contact pair to remain before it dissociates or rearranges a frequency in the decreasing order of I, II ϾϾ III, IV Ͼ V, VI into another contact pair. The first order plots of log (concen- (Table 1). In Ϸ85% of the dimeric states (I and II), the tration of the contact pair) vs. time are shown in Fig. 2, and the Ϫ ϫ 12 Ϫ1 acetate-COO points away from the partner DCE. In these slopes of each plot (kdiss, 10 s ) are summarized in Table 1. 12 Ϫ1 cases, there is little preference in choosing the complexing All of these kdiss are within the range of 10 s . The half-life O O ͞ moiety of DCE ( CH2 or Cl). The slight preference to (log (2) kdiss) of each contact pair is in the decreasing order of Ͼ Ͼ DCE-Cl (I) over DCE-CH2 (II) may be due to the larger surface I, II III, V IV, VI. This finding is comparable to the trend area of the OCl. When acetate-COOϪ is involved in a contact, observed in the relative concentrations of contact pairs at Ͼ Ͼ the preference for the contact is with DCE-CH2 (III, IV) over equilibrium (I, II III, IV V, VI). I and II are the most DCE-Cl (V, VI) due to the negative electrostatic interaction of probable contact pairs and dissociate most slowly. The differ- acetate-COOϪ and DCE-Cl. The atomic charges for the DCE ences between the order of the half-life and the order of methylene C is Ϫ0.06 electronic unit (e.u.), and the charge for concentration at equilibrium are in IV and V. The somewhat OCl is Ϫ0.20 e.u. For IV and VI, it should be noted that the mole factions are obtained by counting contacts involving either O of the carboxylate ion. Thus, the probability to be involved in forming a dimer for each O is the half of the values listed in Table 1. From MD trajectories, one can also study the kinetic stability of each contact pair by calculating the first order rate constants ؊ Table 1. Mole fraction of contact pairs of DCE and CH3CO2 and the first-order rate constants for disappearance of each contact pair 12 Ϫ1 Contact pairs Symbol Mole fraction, % kdiss ϫ 10 ,s OO2CCH3⅐⅐⅐CICH2CH2CI I 43 3.6 OO2CCH3⅐⅐⅐CH2(CI)CH2CI II 41 4.3 Ϫ⅐⅐⅐ H3CCO2 CH2(CI)CH2CI III 6 6.4 Ϫ H3CCOO ⅐⅐⅐CH2(CI)CH2CI IV 623 Ϫ⅐⅐⅐ H3CCO2 CICH2CH2CI V 211 Ϫ H3CCOO ⅐⅐⅐CICH2CH2CI* VI 120 The DCE is present as one of two equivalent gauche conformers.