We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 Å resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the Cα-Cβ-C γ angle (126°) that deviated by 13.4° from the "ideal" bond angle of 112.6°. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C α-Cβ-Cγ angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at Naδ1 and Asp108 in conformation A gave the most exothermic reaction (ΔH = -22 kcal/mol). With His272 doubly protonated at Nδ1 and N ε2, the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 Cα-Cβ-Cγ angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.
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