Non-enzymatic and enzymatic hydrolysis of alkyl halides: A haloalkane dehalogenation enzyme evolved to stabilize the gas-phase transition state of an S _N 2 displacement reaction

Abstract

The semiempirical PM3 method, calibrated against ab initio HF͞6-31؉G(d) theory, has been used to elucidate the reaction of 1,2-dichloroethane (DCE) with the carboxylate of Asp-124 at the active site of haloalkane dehalogenase of Xanthobacter autothropicus. Asp-124 and 13 other amino acid side chains that make up the active site cavity (Glu-56, Trp-125, Phe-128, Phe-172, Trp-175, Leu-179, Val-219, Phe-222, Pro-223, Val-226, Leu-262, Leu-263, and His-289) were included in the calculations. The three most significant observations of the present study are that: (i) the DCE substrate and Asp-124 carboxylate, in the reactive ES complex, are present as an ion-molecule complex with a structure similar to that seen in the gas-phase reaction of AcO ؊ with DCE; (ii) the structures of the transition states in the gasphase and enzymatic reaction are much the same where the structure formed at the active site is somewhat exploded; and (iii) the enthalpies in going from ground states to transition states in the enzymatic and gas-phase reactions differ by only a couple kcal͞mol. The dehalogenase derives its catalytic power from: (i) bringing the electrophile and nucleophile together in a low-dielectric environment in an orientation that allows the reaction to occur without much structural reorganization; (ii) desolvation; and (iii) stabilizing the leaving chloride anion by Trp-125 and Trp-175 through hydrogen bonding.

Functionally relevant motions of haloalkane dehalogenases occur in the specificity-modulating cap domains

“…Requirements for proper flexibility of the catalytic acid could be one of the selection factors responsible for the migration of the catalytic acid in haloalkane dehalogenases (Krooshof et al 1997) and some other ␣/␤-hydrolases . Halide-stabilizing residues of haloalkane dehalogenases are involved in several steps of the catalytic cycle: (1) interact with the halogen atom of the substrate on its binding to a Michaelis-Menten complex (Verschueren et al 1993a), (2) stabilize transition state of the first reaction step (Damborsky et al 1997a;Lightstone et al 1997), and (3) stabilize halide ion released from the substrate during the first reaction step (Verschueren et al 1993a;Damborsky et al 1997a). Dynamic behavior of these residues may influence any of these catalytic steps but may also impact the specificity of dehalogenases toward chlorinated, brominated, and iodinated substrates.…”

Characterization of the Covalent Binding ofN-Phenyl-N′-(2-chloroethyl)ureas to β-Tubulin: Importance of Glu198 in Microtubule Stability

“…The intracellular formation of an ester linkage by xenobiotics such as CEUs is to our knowledge unique. The only prior report of a similar nucleophilic addition of a xenobiotic to a protein under living conditions was the addition of lower alkyl halides, such as 1,2-dichloroethane, on the Asp124 residue of the haloalkane dehalogenase found in Xanthobacter autothropicus (Lightstone et al, 1997). These unexpected results prompted us to evaluate the effect of more potent CEUs on ␤-tubulin and microtubules.…”

Mechanisms and Free Energies of Enzymatic Reactions

“…Thus, the free energy barrier, relative to the ion-dipole complex, for the reaction in the gas phase between acetate ion and DCE can only be 13-14 kcal/mol from the EVB potential, which is markedly different than the barrier height of determined from a variety of high-level ab initio calculations including MP2 and G2 theories. 116,217,[221][222][223] This discrepancy must be somehow absorbed in the parameters of the EVB potential by adjusting the ε 12 term to yield the desired free energy of activation in water. 47 Consequently, such parameter adjustments do not provide solvation effects directly from simulations.…”