Molecular modeling of the reaction pathway and hydride transfer reactions of HMG-CoA reductase

Abstract

HMG-CoA reductase catalyzes the four-electron reduction of HMG-CoA to mevalonate and is an enzyme of considerable biomedical relevance because of the impact of its statin inhibitors on public health. Although the reaction has been studied extensively using X-ray crystallography, there are surprisingly no computational studies that test the mechanistic hypotheses suggested for this complex reaction. Theozyme and quantum mechanical (QM)/molecular mechanical (MM) calculations up to the B3LYP/6-31g(d,p)//B3LYP/6-311++g(2d,2p) level of theory were employed to generate an atomistic description of the enzymatic reaction process and its energy profile. The models generated here predict that the catalytically important Glu83 is protonated prior to hydride transfer and that it acts as the general acid or base in the reaction. With Glu83 protonated, the activation energies calculated for the sequential hydride transfer reactions, 21.8 and 19.3 kcal/mol, are in qualitative agreement with the experimentally determined rate constant for the entire reaction (1 s(-1) to 1 min(-1)). When Glu83 is not protonated, the first hydride transfer reaction is predicted to be disfavored by >20 kcal/mol, and the activation energy is predicted to be higher by >10 kcal/mol. While not involved in the reaction as an acid or base, Lys267 is critical for stabilization of the transition state in forming an oxyanion hole with the protonated Glu83. Molecular dynamics simulations and MM/Poisson-Boltzmann surface area free energy calculations predict that the enzyme active site stabilizes the hemithioacetal intermediate better than the aldehyde intermediate. This suggests a mechanism in which cofactor exchange occurs before the breakdown of the hemithioacetal. Slowing the conversion to aldehyde would provide the enzyme with a mechanism to protect it from solvent and explain why the free aldehyde is not observed experimentally. Our results support the hypothesis that the pK(a) of an active site acidic group is modulated by the redox state of the cofactor. The oxidized cofactor and deprotonated Glu83 are closer in space after hydride transfer, indicating that indeed the cofactor may influence the pK(a) of Glu83 through an electrostatic interaction. The enzyme is able to catalyze the transfer of a hydride to the structurally and electronically distinct substrates by maintaining the general shape of the active site and adjusting the electrostatic environment through acid-base chemistry. Our results are in good agreement with the well-studied hydride transfer reactions catalyzed by liver alcohol dehydrogenase in calculated energy profile and reaction geometries despite different mechanistic functionalities.

Figure 1

Crystal structure PmHMGR/HMG-CoA/NAD+ ternary complex. (pdb code 1QAX)

Figure 2

ONIOM partitioning scheme. QM region is colored by element, link atoms are magenta and the MM region is grey. Hydrogen atoms are hidden for clarity.

Figure 3

Overlay of the PmHMGR active sites of 1QAX (colored by element), neutral Glu83 (yellow), and anionic Glu83 (green). Hydrogen atoms and the main chain atoms for His381 are hidden for clarity.

Figure 4

Theozyme models for the reactant, transition state, and product of the first hydride transfer when Glu83 is not protonated.

Figure 5

Reaction profiles for PmHMGR with the different methods used. Energies calculated for the theozyme models are shown in blue for protonated Glu83 and red for deprotonated Glu83. The ONIOM energies are shown in green.

Figure 6

a) Theozyme and b) ONIOM models for the reactant, transition state, and product of the first hydride transfer when Glu83 is protonated.

Figure 7

a) Theozyme and b) ONIOM models for the reactant, transition state, and product of the second hydride transfer reaction.

Figure 8

a) Binding energies, ΔG_bind, and b) van der Waals energies, ΔG_vdW, from MM/PBSA rescoring of the MD simulations performed for several structures along the model reaction coordinate.

Figure 9

a) Theozyme model of the transition state of hydride transfer to benzaldehyde by LADH b) Comparison of the LADH transition state (left) to the theozyme model hydride transfer to mevaldehyde catalyzed by PmHMGR (right, see Fig.7a) with important distances shown. Hydrogen atoms are hidden for clarity.

Scheme 1

Reactions catalyzed by HMG-CoA Reductase.

Scheme 2

Possible mechanisms for PmHMGR based on the protonation state of Glu83. a) Glu83 is not protonated prior to hydride transfer and Lys267 acts as the proton donor to mevalonate. b) Glu83 is protonated prior to hydride transfer and acts as the proton donor to mevalonate.

Scheme 3

The suggested mechanism from MD simulations and x-ray crystallography for control over the conformation of the first peptide bond by residues Ser85 and His381. a) The conformation observed in MD simulations of the Michaelis complex, where His381 hydrogen bonds to the carbonyl of the first peptide bond and the hydroxyl group of Ser85 is rotated away. b) The conformation observed in the hemidithioacetal crystal structure, where the first peptide bond is rotated 180° and the amide nitrogen is hydrogen bonded to Ser85 while His381 is rotated away or deprotonated to remove its hydrogen bond donor.

Scheme 4

Revised mechanism for PmHMGR.

Scheme 5

The reactions catalyzed by liver alcohol dehydrogenase. a) Aldehyde hydrate oxidation to zinc bound acid b) Alcohol oxidation to aldehyde