Purification and characterization of fully deuterated enzymes

Rokop, S. E.; Gajda, L.; Parmerter, Stanley M.; Crespi, Henry L.; Katz, Joseph

doi:10.1016/0005-2744(69)90365-9

Cited by 34 publications

(22 citation statements)

References 25 publications

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“…In dGST such effects lead to an apparent dimerization of the dimeric units. Rokop et al (1969) measured K m s of protiated and perdeuteriated bacterial alkaline phosphatases and plant ribonucleases and found no significant change on perdeuteriation. Our results for GST were also closely similar.…”

Section: Discussionmentioning

confidence: 99%

“…Our results for GST were also closely similar. Rokop et al (1969) reported an apparent reduction in the rate of catalysis for deuteriated alkaline phosphatase. Interestingly, dGST shows a small (25%) increase in the rate of catalysis relative to pGST; k cat /K m is also apparently increased on deuteriation, which suggests that dGST is actually a (slightly) more efficient enzyme than pGST.…”

Section: Discussionmentioning

confidence: 99%

“…The difference in inactivation temperature between protiated and deuteriated forms of each protein is plotted against the inactivation temperature of the protiated form of each protein. a phycocyanin from Plectonema calothricoides (Hattori et al 1965a, b), b phycocyanin from Phormidium luridum (Hattori et al 1965a, b), c glutathione‐ S ‐transferase from Schistosoma japonicum , d phycocyanin from Synechococcus lividus (Hattori et al 1965a, b), e alkaline phosphatase from Escherichia coli (Rokop et al 1969), f ribonuclease from Fremyella diplosiphon (Rokop et al 1969).…”

Section: Enzyme Kinetics Of Pgst and Dgst—raw Datamentioning

confidence: 99%

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Physicochemical consequences of the perdeuteriation of glutathione S‐transferase from S. japonicum

Brockwell¹

2001

Protein Science

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Glutathione S-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9°C, whereas that for dGST was 51.0°C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K m to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Enzyme Kinetics Of Pgst and Dgst—raw Datamentioning

confidence: 99%

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Physicochemical consequences of the perdeuteriation of glutathione S‐transferase from S. japonicum

Brockwell¹

2001

Protein Science

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“…49, 50 This difference was attributed to the diminution of the vibrational energy available in the active site of the deuterated enzyme. More recently, Schramm and co-workers performed kinetic studies on the human purine nucleoside phosphorylase 51 (PNP) and on the HIV-1 PR.…”

Section: Introductionmentioning

confidence: 99%

Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease

2016

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HIV-1 Protease (HIV-1 PR) is one of the three enzymes essential for the replication process of HIV-1 virus, which explains why it has been the main target for design of drugs against acquired immunodeficiency syndrome (AIDS). This work is focused on exploring the proteolysis reaction catalyzed by HIV-1 PR, with special attention to the dynamic and electrostatic effects governing its catalytic power. Free energy surfaces for all possible mechanisms have been computed in terms of potentials of mean force (PMFs) within hybrid QM/MM potentials, with the QM sub-set of atoms described at semiempirical (AM1) and DFT (M06-2X) level. The results suggest that the most favorable reaction mechanism involves formation of a gem-diol intermediate, whose decomposition into the product complex would correspond to the rate-limiting step. The agreement between the activation free energy of this step with experimental data, as well as kinetic isotope effects (KIEs), supports this prediction. The role of the protein dynamic was studied by protein isotope labelling in the framework of the Variational Transition State Theory. The predicted enzyme KIEs, also very close to the values measured experimentally, reveal a measurable but small dynamic effect. Our calculations show how the contribution of dynamic effects to the effective activation free energy appears to be below 1 kcal·mol−1. On the contrary, the electric field created by the protein in the active site of the enzyme emerges as being critical for the electronic reorganization required during the reaction. These electrostatic properties of the active site could be used as a mould for future drug design.

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“…Yet it is possible to grow microorganisms (e.g. some variants of Escherichia coli) in a highly substituted medium, and achieve almost complete deuterium substitution [5,6].…”

Section: Introductionmentioning

confidence: 99%

Effects of Low-Level Deuterium Enrichment on Bacterial Growth

Xie

Zubarev

2014

PLoS ONE

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Using very precise (±0.05%) measurements of the growth parameters for bacteria E. coli grown on minimal media, we aimed to determine the lowest deuterium concentration at which the adverse effects that are prominent at higher enrichments start to become noticeable. Such a threshold was found at 0.5% D, a surprisingly high value, while the ultralow deuterium concentrations (≤0.25% D) showed signs of the opposite trend. Bacterial adaptation for 400 generations in isotopically different environment confirmed preference for ultralow (≤0.25% D) enrichment. This effect appears to be similar to those described in sporadic but multiple earlier reports. Possible explanations include hormesis and isotopic resonance phenomena, with the latter explanation being favored.

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Purification and characterization of fully deuterated enzymes

Cited by 34 publications

References 25 publications

Physicochemical consequences of the perdeuteriation of glutathione S‐transferase from S. japonicum

Physicochemical consequences of the perdeuteriation of glutathione S‐transferase from S. japonicum

Dynamic and Electrostatic Effects on the Reaction Catalyzed by HIV-1 Protease

Effects of Low-Level Deuterium Enrichment on Bacterial Growth

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