Mechanistic Analysis of Fluorescence Quenching of Reduced Nicotinamide Adenine Dinucleotide by Oxamate in Lactate Dehydrogenase Ternary Complexes

Peng, Huo Lei; Callender, Robert

doi:10.1111/php.12775

Cited by 14 publications

(9 citation statements)

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“…Binding of pyruvate, the substrate, or oxamate, a structural analogue, 23,24 has been observed to quench strongly the cofactor fluorescence by a possible photoinduced electron transfer process. 41,42 Introduction of an inhibitor that binds in the active site, displacing the bound oxamate, would alleviate that quenching. Fig.…”

Section: Resultsmentioning

confidence: 99%

Small molecule cores demonstrate non-competitive inhibition of lactate dehydrogenase

Andrews

Dyer

2018

Med. Chem. Commun.

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Lactate dehydrogenase (LDH) has recently garnered attention as an attractive target for cancer therapies, owing to the enzyme's critical role in cellular metabolism. Current inhibition strategies, employing substrate or cofactor analogues, are insufficiently specific for use as pharmaceutical agents. The possibility of allosteric inhibition of LDH was postulated on the basis of theoretical docking studies of a small molecule inhibitor to LDH. The present study examined structural analogues of this proposed inhibitor to gauge its potency and attempt to elucidate the molecular mechanism of action. These analogues display encouraging inhibition of porcine heart LDH, including micromolar values and a maximum inhibition of up to 50% in the steady state. Furthermore, Michaelis-Menten kinetics and fluorescence data both suggest the simple, acetaminophen derivatives are non-competitive in binding to the enzyme. Kinetic comparisons of a panel of increasingly decorated structural analogues imply that the binding is specific, and the small molecule core provides a privileged scaffold for further pharmaceutical development of a novel, allosteric drug.

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Section: Resultsmentioning

confidence: 99%

Small molecule cores demonstrate non-competitive inhibition of lactate dehydrogenase

Andrews

Dyer

2018

Med. Chem. Commun.

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“…We have studied the affinity of oxamate for two LDH orthologs in the temperature range preceding denaturation. Binding of the substrate to the LDH/NADH binary complex strongly quenches the emission of bound NADH, likely because of interactions with the polar groups of the substrate or its mimic, oxamate, with NADH. − We observed the change in intensity of the 280 and 340 nm peaks (Figure ) to determine isotherms, the association constant, and the free energy for the binding of oxamate to the binary LDH/NADH complex (see Experimental Procedures). van’t Hoff analysis (eq ) in the temperature range of 20–35 °C reveals the association enthalpy of the ternary complexes containing phLDH and cgLDH, and the association entropy was calculated using the Gibbs equation (see Experimental Procedures) (Figure and Table ).…”

Section: Resultsmentioning

confidence: 99%

Thermodynamic and Structural Adaptation Differences between the Mesophilic and Psychrophilic Lactate Dehydrogenases

2017

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The thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (Sus scrofa; a mesophile), and mackerel icefish, cgLDH (Chamapsocephalus gunnari; a psychrophile), were investigated. Using a novel and quite sensitive fluorescence assay that can distinguish protein conformational changes close to and distal from the substrate binding pocket, a reversible global protein structural transition preceding the high-temperature transition (denaturation) was surprisingly found to coincide with a marked change in enzymatic activity for both LDHs. A similar reversible structural transition of the active site structure was observed for phLDH but not for cgLDH. An observed lower substrate binding affinity for cgLDH compared to that for phLDH was accompanied by a larger contribution of entropy to ΔG, which reflects a higher functional plasticity of the psychrophilic cgLDH compared to that of the mesophilic phLDH. The natural osmolyte, trimethylamine N-oxide (TMAO), increases stability and shifts all structural transitions to higher temperatures for both orthologs while simultaneously reducing catalytic activity. The presence of TMAO causes cgLDH to adopt catalytic parameters like those of phLDH in the absence of the osmolyte. Our results are most naturally understood within a model of enzyme dynamics whereby different conformations of the enzyme that have varied catalytic parameters (i.e., binding and catalytic proclivity) and whose population profiles are temperature-dependent and influenced by osmolytes interconvert among themselves. Our results also show that adaptation can be achieved by means other than gene mutations and complements the synchronic evolution of the cellular milieu.

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“…In a previous study, we have shown that oxamate could be an electron acceptor in the quenching of NADH fluorescence when bound to ternary lactate dehydrogenase (LDH) complexes. 16 This prompts us to consider that the quenching of tryptophan fluorescence by oxamate is also a result of photoinduced electron transfer (PET).…”

Section: ■ Introductionmentioning

confidence: 99%

“…Furthermore, as has been shown previously, the correlation between the energies of frontier orbitals of quenchers and Stern−Volmer constants can be established when PET is responsible for the fluorescence quenching. 16 By comparing the frontier orbital energies of quenchers, one then could tell whether a molecule can be a PET donor or acceptor in fluorescence quenching qualitatively or even quantitatively. We apply this concept to the study of the quenching of tryptophan fluorescence by oxamate and pyruvate.…”

Section: ■ Introductionmentioning

confidence: 99%

“…In contrast to this conclusion, we report here that oxamate, a molecule with an amide group and a carboxylate group (nonelectron-withdrawing group) (refer to Figure ), quenches tryptophan fluorescence with a comparable efficiency to acrylamide. In a previous study, we have shown that oxamate could be an electron acceptor in the quenching of NADH fluorescence when bound to ternary lactate dehydrogenase (LDH) complexes . This prompts us to consider that the quenching of tryptophan fluorescence by oxamate is also a result of photoinduced electron transfer (PET).…”

Section: Introductionmentioning

confidence: 99%

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Mechanism for Fluorescence Quenching of Tryptophan by Oxamate and Pyruvate: Conjugation and Solvation-Induced Photoinduced Electron Transfer

Peng

Callender

2018

J. Phys. Chem. B

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Oxamate and pyruvate are isoelectronic molecules. They both quench tryptophan fluorescence with Stern-Volmer constants of 16 and 20 M, respectively, which are comparable to that of arcrylamide, a commonly used probe for protein structure. On the other hand, it is well known that neither the carboxylate group of these molecules nor the amide group is a good quencher. To find the mechanism of the quenching by oxamate and pyruvate, density functional theory computations with a polarizable continuum model, solvation based on density, and explicit waters, were performed. Results indicate that both molecules can be an electron acceptor via photoinduced electron transfer. There are two requirements. First, the carboxylate and amide moieties must be in direct contact to bring about noticeable quenching. The conjugation between the amide (or the keto) group and the carboxylate group leads to a lower π* orbital, which is the lowest unoccupied molecular orbital (LUMO), and can then accept an electron from the excited tryptophan. Second, since oxamate and pyruvate ions have high electron density, hydrogen bonds with waters, which can be simulated by an explicit water model, are essential. Their LUMO energies are strongly influenced by water in aqueous solution. The above findings demonstrate how tryptophan fluorescence gets quenched in aqueous solution. The findings may be important in dealing with those problems where frontier orbitals are considered, especially with molecules having high electron density.

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Mechanistic Analysis of Fluorescence Quenching of Reduced Nicotinamide Adenine Dinucleotide by Oxamate in Lactate Dehydrogenase Ternary Complexes

Cited by 14 publications

References 100 publications

Small molecule cores demonstrate non-competitive inhibition of lactate dehydrogenase

Small molecule cores demonstrate non-competitive inhibition of lactate dehydrogenase

Thermodynamic and Structural Adaptation Differences between the Mesophilic and Psychrophilic Lactate Dehydrogenases

Mechanism for Fluorescence Quenching of Tryptophan by Oxamate and Pyruvate: Conjugation and Solvation-Induced Photoinduced Electron Transfer

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