25 research outputs found
Supporting data for characterization of the busulfan metabolite EdAG and the Glutaredoxins that it adducts
This article describes data related to a research article titled âThe Busulfan Metabolite EdAG Irreversibly Glutathionylates Glutaredoxinsâ [1]. EdAG is an electrophilic GSH analog formed in vivo from busulfan, which is used in hematopoietic stem cell transplants. EdAG glutathionylates Glutaredoxins (Grx's) but not glutathione transferase A1-1 (GSTA1-1) in vitro. This article includes a complete NMR characterization of synthetic EdAG including homonuclear and heteronuclear correlation spectra. Also included are mass spectra of peptides from Grx's or GSTA1-1 that have cys residues that do not react with EdAG
Reversibility and Low Commitment to Forward Catalysis in the Conjugation of Lipid Alkenals by Glutathione Transferase A4-4
High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for âforwardâ Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict âone wayâ detoxication enzyme
Reversibility and Low Commitment to Forward Catalysis in the Conjugation of Lipid Alkenals by Glutathione Transferase A4-4
High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for ‘forward’ Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict ‘one way’ detoxication enzyme
Reaction Dynamics of ATP Hydrolysis Catalyzed by PâGlycoprotein
P-glycoprotein
(P-gp) is a member of the ABC transporter family
that confers drug resistance to many tumors by catalyzing their efflux,
and it is a major component of drugâdrug interactions. P-gp
couples drug efflux with ATP hydrolysis by coordinating conformational
changes in the drug binding sites with the hydrolysis of ATP and release
of ADP. To understand the relative rates of the chemical step for
hydrolysis and the conformational changes that follow it, we exploited
isotope exchange methods to determine the extent to which the ATP
hydrolysis step is reversible. With Îł<sup>18</sup>O<sub>4</sub>-labeled ATP, no positional isotope exchange is detectable at the
bridging β-phosphorusâOâÎł-phosphorus bond.
Furthermore, the phosphate derived from hydrolysis includes a constant
ratio of three <sup>18</sup>O/two <sup>18</sup>O/one <sup>18</sup>O that reflects the isotopic composition of the starting ATP in multiple
experiments. Thus, H<sub>2</sub>O-exchange with HPO<sub>4</sub><sup>2â</sup> (P<sub>i</sub>) was negligible, suggesting that a
[P-gp¡ADP¡P<sub>i</sub>] is not long-lived. This further
demonstrates that the hydrolysis is essentially irreversible in the
active site. These mechanistic details of ATP hydrolysis are consistent
with a very fast conformational change immediately following, or concomitant
with, hydrolysis of the Îł-phosphate linkage that ensures a high
commitment to catalysis in both drug-free and drug-bound states
Gas-Phase Hydrogen/Deuterium Exchange for Distinguishing Isomeric Carbohydrate Ions
The structural diversity of carbohydrates
presents a major challenge
for glycobiology and the analysis of glycoconjugates. Mass spectrometry
has become a primary tool for glycan analysis thanks to its speed
and sensitivity, but the information content regarding the glycan
structure of protonated glycoconjugates is hindered by the inability
to differentiate linkage and stereoisomers. Here, we examine a variety
of protonated carbohydrate structures by gas-phase hydrogen/deuterium
exchange (HDX) to discover that the exchange rates are distinct for
isomeric carbohydrates with even subtle structural differences. By
incorporating an internal exchange standard, HDX could effectively
distinguish all linkage and stereoisomers that were examined and presents
a mass spectrometry-based approach for glycan structural analysis
with immense potential
Backbone dynamics of human parathyroid hormone (1-34): Flexibility of the central region under different environmental conditions
The presence of a stable tertiary structure in the bioactive N-terminal portion of parathyroid hormone (PTH), a major hormone in the maintenance of extracellular calcium homeostasis, is still debated. In this work, 15N relaxation parameters of the 33 backbone amides of human PTH(1\u201334) were determined in phosphate-buffered saline solution (PBS) and in the presence of dodecylphosphocholine (DPC) micelles. The relaxation parameters were analyzed using both the model-free formalism (G. Lipari and A. Szabo, Journal of the American Chemical Society, 1982, Vol. 104, pp. 4546\u20134549) and the reduced spectral density functions approach (J.-F. Lefevre, K. T. Dayie, J. W. Peng, and G. Wagner, Biochemistry, 1996, Vol. 35, pp. 2674\u20132686). In PBS, the region around Gly12 possesses a high degree of flexibility and the C-terminal helix is less flexible than the N-terminal one. In the presence of DPC micelles, the mobility of the entire molecule is reduced, but the stability of the N-terminal helix increases relative to the C-terminal one. A point of relatively higher mobility at residue Gly12 is still present and a new site of local mobility at residues 16\u201317 is generated. These results justify the lack of experimental nuclear Overhauser effect (NOE) restraints with lack of tertiary structure and support the hypothesis that, in the absence of the receptor, the relative spatial orientation of the two N- and C-terminal helices is undefined. The flexibility in the midregion of PTH(1\u201334), maintained in the presence of the membrane-mimetic environment, may enable the correct relative disposition of the two helices, favoring a productive interaction with the receptor