QM/MM (ONIOM) Study of Glycerol Binding and Hydrogen Abstraction by the Coenzyme B₁₂-Independent Dehydratase

Abstract

Glycerol binding and the radical-initiated hydrogen transfer by the coenzyme B(12)-independent glycerol dehydratase from Clostridium butyricum were investigated by using quantum mechanical/molecular mechanical (QM/MM) calculations based on the high-resolution crystal structure (PDB code: 1r9d). Our QM/MM calculations of enzyme catalysis considered the electrostatic coupling between the quantum-mechanical and molecular-mechanical subsystems and two alternative mechanisms. In addition to performing QM/MM calculations in the enzyme, we evaluated energetics along the same reaction pathway in aqueous solution modeled by the polarized dielectric and in the virtual enzyme site that included full steric component from the enzyme residues described by molecular mechanics but lacked the electrostatic contribution of these residues. In this way, we established significant enzyme catalytic effect with respect to reference reactions in both an aqueous solution and a nonpolar cavity. Structurally, four hydrogen bonds formed between glycerol and H164, S282, E435, and D447 anchor glycerol for hydrogen abstraction by thiyl radical on C433. These hydrogen-bond partners orient glycerol molecule to facilitate the formation of the transition state for hydrogen abstraction from carbon C1. This reaction then proceeds with the activation free energy of 6.3 kcal/mol and the reaction free energy of 6.1 kcal/mol. The polarization effects imposed by these hydrogen bonds represent a predominant contribution to a 7.5 kcal/mol enzyme catalytic effect. These results demonstrate the importance of electrostatic catalysis and hydrogen-bonding in enzyme-catalyzed radical reactions and advance our understanding of the catalytic mechanism of B(12)-independent glycerol dehydratases.

Glycerol as a Substrate and Inactivator of Coenzyme B12‐Dependent Diol Dehydratase

“…In the context of kinetics, the energy barriers for hydrogen abstraction from either C1 or C3 are similar (58.4 and 52.0 kJ mol −1 for TS1 C1 and TS1 C3 , respectively). Ullman and Liu obtained a somewhat lower activation barrier for H‐atom abstraction from C1 than from C3; however, they used a different computational approach [27,30] . After H‐atom abstraction from C1, a direct elimination of water from the substrate‐derived radical R .…”

“…A parallel investigation will examine the analogous scenario with PDO as the substrate. Finally, since it was shown that H-atom abstraction from C3 is possible in B 12 -iGDH, [27,30] we explored a pathway that begins with H-atom abstraction from C3 in this enzyme too, in an effort to develop a comprehensive description of inactivation processes in coenzyme B 12 -dependent and coenzyme B 12 -independent enzymes.…”

“…Ullman and Liu obtained a somewhat lower activation barrier for H-atom abstraction from C1 than from C3; however, they used a different computational approach. [27,30] After H-atom abstraction from C1, a direct elimination of water from the substrate-derived radical R * C1 occurs. [25] This elimination is facilitated by proton transfer from Lys323 via His281 to C2À OH of GOL and is accompanied by deprotonation of C1À OH by the residue Glu435; this process is similar to the previously mentioned "push-pull" effect that occurs in B 12 -dDDH catalysis.…”

Reversible Hydrogen Transfer Reactions in Thiyl Radicals From Cysteine and Related Molecules: Absolute Kinetics and Equilibrium Constants Determined by Pulse Radiolysis

“…There is increasing evidence that thiyl radicals play an important role in enzyme-catalyzed transformations, synthetic organic chemistry, and the post-translational modification of proteins. 1–4 For example, ribonucleotide reductase and pyruvate–formate lyase utilize CysS • radicals for hydrogen abstraction and addition reactions, respectively. Here, reversible hydrogen transfer reactions between radicals and C–H and S–H bonds of glycine (Gly) and Cys, respectively, provide CysS • radicals in a protein conformation-dependent pathway.…”

Cysteine residues as catalysts for covalent peptide and protein modification: a role for thiyl radicals?

“…Hence, for aliphatic alcohols and ethers, equilibrium 7 is located far on the left hand side, well accounted for by the differences in the homolytic bond dissociation energies (BDE) between the S-H bond of Cys, BDE(S-H, Cys) = 367 kJ/mol [13], and the C-H bonds of aliphatic alcohols and ethers, e.g., BDE(C-H) = 393 kJ/mol for CH 3 OH [14]. Nevertheless, several enzymes utilize thiyl radicals for turnover: in ribonucleotide reductase [15-17], benzylsuccinate synthase [18, 19], and glycerol dehydratase [20], thiyl radicals engage in hydrogen transfer reactions with substrates (while in pyruvate formate lyase, thiyl radicals add to the carbonyl group of pyruvate [17, 21]). $${\mathrm{RS}}^{•}+\text{-}\mathrm{O}\text{-}\mathrm{CH}\left(\mathrm{normalR}’\right)\text{-}\rightleftharpoons \mathrm{RSH}+\text{-}\mathrm{O}\text{-}{\mathrm{normalC}}^{•}\left(\mathrm{normalR}’\right)\text{-}$$…”