The Organization of Active Site Side Chains of Glycerol-3-phosphate Dehydrogenase Promotes Efficient Enzyme Catalysis and Rescue of Variant Enzymes

Cristobal, Judith R.; Reyes, Archie C.; Richard, John P.

doi:10.1021/acs.biochem.0c00175

Cited by 12 publications

(32 citation statements)

References 41 publications

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“… 11 , 20 There is similar evidence that binding interactions of exogenous phosphite dianion and of ethyl ammonium cations, respectively, are utilized to stabilize active closed conformations of wild-type hl GPDH complexed to GA and of the K120A variant complexed to DHAP. 68 These results emphasize the importance of the utilization of the substrate or side chain binding energy in the stabilization of rigid and structured active enzymes. 69 , 70 It is not surprising that it might be difficult to model effects of side chain substitutions that promote a large relaxation of the tight MC to an open protein.…”

Section: Resultsmentioning

confidence: 85%

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

et al. 2020

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Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition-state stabilization. While the human form ( hl GPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hl GPDH and its variants, as well as providing a crystal structure of the binary hl GPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted hl GPDH and investigated the effect of mutations on catalysis from a detailed structural study. In particular, the K120A and R269A variants increase both the volume and solvent exposure of the active site, with concomitant loss of catalytic activity. In addition, the R269 side chain interacts with both the Q295 side chain on the catalytic loop, and the substrate phosphodianion. Our structural data and simulations illustrate the critical role of this side chain in facilitating the closure of hl GPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297). This, in turn, rationalizes a tremendous 41,000 fold decrease experimentally in the turnover number, k cat , upon truncating this residue, as loop closure is essential for both correct positioning of key catalytic residues in the active site, as well as sequestering the active site from the solvent. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of allosteric inhibitors targeting this biomedically important enzyme.

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Section: Resultsmentioning

confidence: 85%

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

et al. 2020

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“…11,20 There is similar evidence that binding interactions of exogenous phosphite dianion and of ethyl ammonium cation, respectively, are utilized to stabilize active closed conformations of wild-type hlGPDH complexed to glycoaldehyde, and of the K120A variant complexed to DHAP. 78 These results emphasize the importance of the utilization of substrate or side chain binding energy in the stabilization of rigid and structured active enzymes. 79,80 It is not surprising that it might be difficult to model effects of side-chain substitutions that promote a large relaxation of the tight Michaelis complex to an open protein.…”

mentioning

confidence: 84%

“…The ion pair between the K120 and D260 side chains at wild-type hlGPDH, suggests a role for D260 in (pre)organization of the K120 side chain to provide optimal stabilizing interactions with the anionic transition state. 5,78 The K120A substitution replaces stabilizing interactions between the side chain cation and anionic transition state with destabilizing interactions from the ionized D260 side chain anion. The K120A substitution results in a calculated increase in the pKa of the D260 side chain from 4.0±0.003 to 6.0±0.004 (average values and standard error of the mean of pKa values estimated for snapshots taken every 100 ps of our equilibration runs, evaluated using PROPKA 3.1 34 ).…”

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confidence: 99%

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

Mhashal¹,

Romero‐Rivera²,

Mydy³

et al. 2020

Preprint

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<div> <div> <div> <p>Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition state stabilization. While the human form (hlGPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend the experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hlGPDH and its variants, as well as providing a novel crystal structure of the binary hlGPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted variants of hlGPDH and investigated the effect of mutations on the catalysis from a detailed structural study. Our structural data and simulations also illustrate the critical role of the R269 side chain in facilitating the closure of hlGPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297), thus rationalizing a tremendous 41,000-fold decrease experimentally in the turnover number, kcat, upon truncating this residue. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of novel allosteric inhibitors targeting this biomedically important enzyme.</p></div></div></div>

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“…The ability of enzymes to bind and stabilize the transition state has been analyzed from several perspectives (see for example, References 7,25,58) . 56 Two other differences that distinguish the GPDH measurement from others in the table are that the proton transfer occurs after the catalytic step 57 (hydride anion transfer) and is not rate-limiting, and it involves acid catalysis rather than base catalysis. barrier.…”

Section: Transition State Effects: Substrate Bindingmentioning

confidence: 99%

How enzymes harness highly unfavorable proton transfer reactions

Silverstein

2021

Protein Science

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Acid-base reactions that are exceedingly unfavorable under standard conditions can be catalytically important at enzyme active sites. For example, in triose phosphate isomerase, a glutamate side chain (nominal pK a ≈ 4 in solution) can in fact deprotonate a CH group that is vicinal to a carbonyl (pK a ≈ 18 in solution). This is true because of three distinct interactions: (a) ground state pK a shifts due to environment polarity and electrostatics; (b) dramatic increases in effective molarity due to optimization of proximity and orientation; and (c) transition state pK a shifts due to binding interactions and the formation of strong low barrier hydrogen bonds. In this report, we review the literature showing that the sum of these three effects supplies more than enough free energy to push forward proton transfer reactions that under standard conditions are exceedingly nonspontaneous and slow.

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The Organization of Active Site Side Chains of Glycerol-3-phosphate Dehydrogenase Promotes Efficient Enzyme Catalysis and Rescue of Variant Enzymes

Cited by 12 publications

References 41 publications

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-phosphate Dehydrogenase

Modeling the Role of a Flexible Loop and Active Site Side Chains in Hydride Transfer Catalyzed by Glycerol-3-Phosphate Dehydrogenase

How enzymes harness highly unfavorable proton transfer reactions

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