HMG-CoA reductase catalyzes the four electron reduction of HMG-CoA to mevalonate and is an enzyme of considerable biomedical relevance due to 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 QM/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/base in the reaction. With Glu83 protonated, the activation energy 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/min). When Glu83 is not protonated, the first hydride transfer reaction is predicted to be disfavored by over 20 kcal/mol, and the activation energy is predicted to be higher by over 10 kcal/mol. While not involved in the reaction as an acid/base, Lys267 is critical for stabilization of the transition state in forming an oxyanion hole with the protonated Glu83. Molecular dynamics simulations and MM/PBSA free energy calculations predict that the enzyme active site stabilizes the hemithioacetal intermediate better than the aldehyde intermediate. This suggests a mechanism where 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 pKa of an active site acidic group is modulated by the redox state of the cofactor. The oxidized cofactor and deprotonated Glu83 get closer after hydride transfer indicating that indeed the cofactor may influence the pKa of Glu83 through an electrostatic interaction. The enzyme is able to catalyze hydride transfer 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 (LADH) in calculated energy profile and reaction geometries despite different mechanistic functionalities.

CONSPECTUS The seven decades of research on the mechanism of HMG-CoA Reductase (HMGR) provided a detailed reaction pathway for what is one of the most biomedically important and mechanistically most complex enzymes. HMGR is the target of statins that are prescribed to improve the quality of life of millions of people worldwide and, more recently, has been identified as a target for the development of antimicrobial agents. The impact of advances in diverse research areas, such as molecular biology and computational chemistry, are reflected in the maturation of mechanistic proposals for HMGR. Thus, the development of state-the-art methods in enzyme mechanism research can be traced by following the development of the HMGR mechanism. Similarly, the questions raised about these mechanism proposals reflect the limitations of these methods. The mechanism of HMGR, a four-electron oxidoreductase, has been uncovered to be unique and far more complex than originally thought. The reaction contains multiple chemical steps, coupled to large- scale domain motions of the homodimeric enzyme. The first proposals for the HMGR mechanism were based on kinetic and labeling experiments, drawing analogies to the mechanism of known dehydrogenases. Advances in molecular biology and bioinformatics enabled site-directed mutagenesis experiments and protein sequence analysis which identified catalytically important glutamate, aspartate, and histidine residues that in turn generated new and more complicated mechanistic proposals. With the development of protein crystallography, HMGR crystal structures were solved to reveal the spatial organization of the active site with an unexpected lysine residue lying at its center. The multitude of crystal structures led to more and more complex mechanistic proposals but the inherent limitations of the protein crystallography left a number of questions unresolved. For example, the proposed mechanisms change based on the protonation state of the active site glutamate residue, which cannot be clearly determined from the crystal structures. As computational analysis of large biomolecules become more feasible, application of methods such as hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to the HMGR mechanism have led to the most detailed mechanistic proposal yet. As these methodologies continue to improve, their power to study enzyme mechanism in conjunction with protein crystallography is enormous. Nevertheless, even the most current mechanistic proposal is yet incomplete due to limitations of the current computational methodologies. Thus, HMGR serves as a model for how combination of increasingly sophisticated experimental and computational methods can elucidate very complex enzyme mechanisms.

The macrocyclic depsipeptide Largazole is a potent inhibitor of metal-dependent histone deacetylases (HDACs), some of which are drug targets for cancer chemotherapy. Indeed, Largazole partially resembles Romidepsin (FK228), a macrocyclic depsipeptide already approved for clinical use. Each inhibitor contains a pendant side chain thiol that coordinates to the active site Zn2+ ion, as observed in the X-ray crystal structure of the HDAC8–Largazole complex [Cole, K. E.; Dowling, D. P.; Boone, M. A.; Phillips, A. J.; Christianson, D. W. J. Am. Chem. Soc. 2011, 133, 12474]. Here, we report the X-ray crystal structures of HDAC8 complexed with three synthetic analogues of Largazole in which the depsipeptide ester is replaced with a rigid amide linkage. In two of these analogues, a 6-membered pyridine ring is also substituted (with two different orientations) for the 5-membered thiazole ring in the macrocycle skeleton. The side chain thiol group of each analogue coordinates to the active site Zn2+ ion with nearly ideal geometry, thereby preserving the hallmark structural feature of inhibition by Largazole. Surprisingly, in comparison with the binding of Largazole, these analogues trigger alternative conformational changes in the L1 and L2 loops flanking the active site. However, despite these structural differences, inhibitory potency is generally comparable to, or just moderately less than, the inhibitory potency of Largazole. Thus, this study reveals important new structure-affinity relationships for the binding of macrocyclic inhibitors to HDAC8.

C–H activation, C–H functionalization, cyclopalladation, mono-protected amino acid, dimeric Pd amino acid complexes, MPAA coordination, relay of stereochemistry.