Kinetic analysis of the individual reductive steps catalyzed by β-hydroxy-β-methylglutaryl-coenzyme A reductase obtained from yeast

Qureshi, Nilofer; Dugan, Richard E.; Cleland, W. W.; Porter, John W.

doi:10.1021/bi00664a010

Cited by 45 publications

(40 citation statements)

References 14 publications

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“…Results from kinetic experiments and absence of the free aldehyde are consistent with the hypothesis that the cofactor exchange step occurs before breakdown of the hemithioacetal. 23–26 MM/PBSA free energy calculations indicate that the enzyme active site stabilizes the hemithioacetal relative to the aldehyde. Consequently, the opening/closing process of the flap domain and cofactor exchange must be faster than hemithioacetal breakdown after the thioester is reduced.…”

Section: Discussionmentioning

confidence: 99%

Molecular Modeling of the Reaction Pathway and Hydride Transfer Reactions of HMG-CoA Reductase

et al. 2012

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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.

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Section: Discussionmentioning

confidence: 99%

Molecular Modeling of the Reaction Pathway and Hydride Transfer Reactions of HMG-CoA Reductase

et al. 2012

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“…Most research was performed on the yeast HMGR, which had been used to study the curious absence of the aldehyde in free form during the reaction that was inconsistent with the proposed reaction pathway and to explore the possibility of a hemithioacetal intermediate. 31,34,35,42 Qureshi et al . described the stereoselective chemical mechanism of hydride transfer and hemithioacetal breakdown, implicating unspecified acidic and basic residues.…”

Section: The Early Hmgr Mechanism Proposals and The Discovery Of Pmhmgrmentioning

confidence: 99%

“…described the stereoselective chemical mechanism of hydride transfer and hemithioacetal breakdown, implicating unspecified acidic and basic residues. 34 Shortly afterwards, Veloso et al . expanded the mechanism using pH dependent kinetic analyses to specify an acidic residue and a cationic histidine as the catalytic residues.…”

Section: The Early Hmgr Mechanism Proposals and The Discovery Of Pmhmgrmentioning

confidence: 99%

The Increasingly Complex Mechanism of HMG-CoA Reductase

2013

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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.

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“…This thiohemiacetal would then be in equilibrium with the reduced CoA and mevaldehyde, the aldehyde thought to be the active substrate for the second reduction reaction. 7 The NAD + is released to solution in preparation for binding of another NADH for the second hydride transfer that converts the aldehyde to an alcohol, mevalonate.…”

mentioning

confidence: 99%

A Novel Role for Coenzyme A during Hydride Transfer in 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

et al. 2013

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In this study, we take advantage of the ability of HMG-CoA reductase (HMGR) from Pseudomonas mevalonii to remain active while in its crystallized form to study the changing interactions between the ligands and protein as the first reaction intermediate is created. HMG-CoA reductase catalyzes one of the few double oxidation–reduction reactions in intermediary metabolism that take place in a single active site. Our laboratory has undertaken an exploration of this reaction space using structures of HMG-CoA reductase complexed with various substrate, nucleotide, product, and inhibitor combinations. With a focus in this publication on the first hydride transfer, our structures follow this reduction reaction as the enzyme converts the HMG-CoA thioester from a flat sp2-like geometry to a pyramidal thiohemiacetal configuration consistent with a transition to an sp3 orbital. This change in the geometry propagates through the coenzyme A (CoA) ligand whose first amide bond is rotated 180° where it anchors a web of hydrogen bonds that weave together the nucleotide, the reaction intermediate, the enzyme, and the catalytic residues. This creates a stable intermediate structure prepared for nucleotide exchange and the second reduction reaction within the HMG-CoA reductase active site. Identification of this reaction intermediate provides a template for the development of an inhibitor that would act as an antibiotic effective against the HMG-CoA reductase of methicillin-resistant Staphylococcus aureus.

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Kinetic analysis of the individual reductive steps catalyzed by β-hydroxy-β-methylglutaryl-coenzyme A reductase obtained from yeast

Cited by 45 publications

References 14 publications

Molecular Modeling of the Reaction Pathway and Hydride Transfer Reactions of HMG-CoA Reductase

Molecular Modeling of the Reaction Pathway and Hydride Transfer Reactions of HMG-CoA Reductase

The Increasingly Complex Mechanism of HMG-CoA Reductase

A Novel Role for Coenzyme A during Hydride Transfer in 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase

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