Enzymology at Subzero Temperatures

Douzou, Pierre

doi:10.1002/9780470122907.ch3

Cited by 20 publications

(6 citation statements)

References 89 publications

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“…The methods of pH measurements and the properties of the most popular buffer systems in mixtures of water with alcohols at subzero temperatures have been studied and reviewed by Douzou and colleagues (29,36,41). Despite the fact that some buffers, such as acetate and phosphate have much smaller pK a temperature dependence than amine buffers, a general recommendation for the most stable buffer system is difficult because of the changes of ionization enthalpies of these groups in organic and mixed solvents.…”

Section: Introductionmentioning

confidence: 99%

“…Despite the fact that some buffers, such as acetate and phosphate have much smaller pK a temperature dependence than amine buffers, a general recommendation for the most stable buffer system is difficult because of the changes of ionization enthalpies of these groups in organic and mixed solvents. Several reviews by Douzou provide tabulated reference data for several buffers in mixed aqueous – organic solvents (24, 29, 36). …”

Section: Introductionmentioning

confidence: 99%

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Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

Denisov

Grinkova

Sligar

2012

Methods in Molecular Biology

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Cryogenic radiolytic reduction is one of the most simple and convenient methods of generation and stabilization of reactive iron-oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxoferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

Denisov

Grinkova

Sligar

2012

Methods in Molecular Biology

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“…One can perturb by changing the experimental conditions. Cryoenzymology is an example of this technique which involves two perturbants: temperature and antifreeze . We have exploited cryoenzymology with yPGK by including methanol in the reaction medium and working at 4 °C, and by this means, we were able to build a seven-step reaction pathway and to obtain estimates for its rate constants , .…”

mentioning

confidence: 99%

Direct Kinetic Evidence That Lysine 215 Is Involved in the Phospho-Transfer Step of Human 3-Phosphoglycerate Kinase

et al. 2009

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3-Phosphoglycerate kinase (PGK) is a promising candidate for the activation of nucleotide analogues used in antiviral and anticancer therapies. PGK is a key enzyme in glycolysis; it catalyzes the reversible reaction 1,3-bisphosphoglycerate + ADP <--> 3-phosphoglycerate + ATP. Here we explored the catalytic role in human PGK of the highly conserved Lys 215 that has been proposed to be essential for PGK function by a transient and equilibrium kinetic study with the active site mutant K215A. By the stopped-flow method we show that the kinetics of substrate binding and the associated protein isomerization steps are fast and identical for the wild-type PGK and mutant K215A. By the use of a chemical sampling method (rapid quench flow) under multiple and single turnover conditions and in both directions of the reaction, we show that the rate-limiting step with wild-type PGK follows product formation (presumably product release), whereas with the mutant it is the phospho-transfer step itself that is rate-limiting. Mutant K215A has a low inherent phosphotransferase activity, and to explain this, we carried out a molecular modeling study. This suggests that with the mutant the conserved Arg 65 replaces the missing Lys 215 by helping to position the transferable phospho group during the reaction. Molecular dynamics simulations suggest that in the mutant the closed conformation of the enzyme is stabilized by a salt bridge between Asp 218 and Arg 170 rather than Arg 65 in the wild-type PGK.

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“…Investigation of proteins in aqueous environments at subzero temperatures has attracted significant attention in research areas ranging from cryobiochemistry and cryoenzymology, [1][2][3] protein dynamics, 4 and protein cold denaturation 5,6 to the study of cellular water. 7 These types of experiments present challenges because they require one to create a low temperature fluid environment and simultaneously prevent samples from freezing at the sub-zero temperatures.…”

Section: Introductionmentioning

confidence: 99%

“…7 These types of experiments present challenges because they require one to create a low temperature fluid environment and simultaneously prevent samples from freezing at the sub-zero temperatures. One can maintain fluidity by using additives, such as acetone, dimethyl formamide, glycerol, or methanol, instead of pure water or aqueous buffers; [1][2][3] however, these mixtures are not entirely satisfactory mimics of biological environments. Narrow-bore capillaries have also been used to prevent nucleation and ice formation.…”

Section: Introductionmentioning

confidence: 99%

On the Behavior of Indole-Containing Species Sequestered within Reverse Micelles at Sub-Zero Temperatures

Munson

Kelepouris

Baker³

et al. 2007

Appl Spectrosc

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We report on the effects of temperature (+30 to -100 degrees C) on the fluorescence from N-acetyl tryptophanamide (NATA) and human serum albumin (HSA) sequestered within Aerosol-OT (AOT) reversed micelles. NATA reports simultaneously from the polar and non-polar side of the reverse micelle interface. As the sample temperature decreases, the relative fraction of NATA molecules associated with the polar side increases. This redistribution process is characterized by DeltaH = -14.8 +/- 0.6 kJ/mol and DeltaS = -54 +/- 2 J/(K mol). The activation energy for thermal quenching (E(a,TQ)) associated with the polar side NATA molecules is 6.7 kJ/mol before the micelles have shed water and 1.0 kJ/mol after water shedding (below approximately -20 degrees C). The time-resolved fluorescence intensity decay for tryptophan-214 in HSA is triple exponential. We suggest that these lifetimes arise from three indole residue conformations in equilibrium. Cooling the sample causes a freezing-in of the least quenched conformer; the other conformers are frozen out. The E(a,TQ) value for the shortest lifetime component is 6 kJ/mol. The E(a,TQ) for the long and intermediate lifetime components are equivalent (approximately 1.5 kJ/mol).

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Enzymology at Subzero Temperatures

Cited by 20 publications

References 89 publications

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

Direct Kinetic Evidence That Lysine 215 Is Involved in the Phospho-Transfer Step of Human 3-Phosphoglycerate Kinase

On the Behavior of Indole-Containing Species Sequestered within Reverse Micelles at Sub-Zero Temperatures

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