Human glyoxalase II contains an Fe(II)Zn(II) center but is active as a mononuclear Zn(II) enzyme.
ABSTRACT Human glyoxalase II (Glx2) was overexpressed in rich medium and in minimal medium containing zinc, iron, or cobalt, and the resulting Glx2 analogues were characterized using metal analyses, steady-state and pre-steady-state kinetics, and NMR and EPR spectroscopies to determine the nature of the metal center in the enzyme. Recombinant human Glx2 tightly binds nearly 1 equiv each of Zn(II) and Fe. In contrast to previous reports, this study demonstrates that an analogue containing 2 equiv of Zn(II) cannot be prepared. EPR studies suggest that most of the iron in recombinant Glx2 is Fe(II). NMR studies show that Fe(II) binds to the consensus Zn(2) site in Glx2 and that this site can also bind Co(II) and Ni(II), suggesting that Zn(II) binds to the consensus Zn(1) site. The NMR studies also reveal the presence of a dinuclear Co(II) center in Co(II)-substituted Glx2. Steady-state and pre-steady-state kinetic studies show that Glx2 containing only 1 equiv of Zn(II) is catalytically active and that the metal ion in the consensus Zn(2) site has little effect on catalytic activity. Taken together, these studies suggest that Glx2 contains a Fe(II)Zn(II) center in vivo but that the catalytic activity is due to Zn(II) in the Zn(1) site.
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ABSTRACT: The mitochondrial pool of GSH (glutathione) is considered the major redox system in maintaining matrix redox homeostasis, preserving sulfhydryl groups of mitochondrial proteins in appropriate redox state, protecting mitochondrial DNA against mitochondrial-derived ROS and in defending mitochondrial membranes against oxidative damage. Despite its importance in maintaining mitochondrial functionality, GSH is synthesized exclusively in the cytoplasm and must be actively transported into mitochondria. In this work we found that SLG (S-D-Lactoylglutathione), an intermediate of the glyoxalase system, can enter the mitochondria and there be hydrolyzed from mitochondrial glyoxalase II enzyme to D-lactate and GSH. We demonstrated SLG transport from cytosol to mitochondria by incubating substrates with radioactive compounds that showed two different kinetic curves for SLG or GSH substrates, indicating different kinetic transport. The incubation of functionally and intact mitochondria with SLG showed increased GSH levels in normal mitochondria and in artificially uncoupled mitochondria demonstrating transport not linked to ATP presence. Also mitochondrial-swelling assay confirmed SLG entrance into organelles. Moreover we observed oxygen uptake and generation of membrane potential probably linked to D-lactate oxidation which is a product of SLG hydrolysis. The latter data was confirmed by oxidation of D-lactate in mitochondria evaluated by measuring mitochondrial D-lactate dehydrogenize activity. In this work we also showed the presence of mitochondrial glyoxalase II, the enzyme that catalyzes SLG hydrolysis, in inter-membrane space and mitochondrial matrix. In conclusion, this work showed new alternative sources of GSH supply to the mitochondria by SLG, an intermediate of the glyoxalase system.Free Radical Biology and Medicine 12/2013; 67. DOI:10.1016/j.freeradbiomed.2013.12.005
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ABSTRACT: We introduce the concept of 'chiral compartmentation' in metabolism that emerges from the stereo-specificity of enzymes for their substrate(s). The fully differentiated mammalian erythrocyte has no sub-cellular organelles and yet it displays compartmentation of lactic acid that is generated either by glycolysis or the glyoxalase pathway. A form of 'operational compartmentation' exists, based not on the chemistry of the reactive groups in the molecules but their stereoisomerism. This we call 'chiral compartmentation', and the rationale for its 'natural selection' in the erythrocyte (and presumably in the cytoplasm of other cells) is discussed. Increasing awareness of the presence of d-amino acids in proteins in the otherwise dominant 'l-chiral biosphere', and of the preferential use of one enantiomer of a metabolite versus the other is largely due to recent developments in rapidly-applicable, analytical-chemical methods. We confirmed that the glyoxalase pathway yields d-lactic acid by using nuclear magnetic resonance (NMR) spectroscopy of stretched chiral hydrogels. The activity of the two lactate-producing pathways have been described by numerical integration of simultaneous non-linear differential equations, based on enzyme models like that introduced by Michaelis and Menten in 1913.FEBS letters 05/2013; 587(17). DOI:10.1016/j.febslet.2013.05.025
Conference Paper: A 15-bit 2 MHz Nyquist rate ΔΣ ADC in a 1 µm CMOS technology[Show abstract] [Hide abstract]
ABSTRACT: A high-resolution high-speed fourth order cascaded delta-sigma modulator, based on a 2-1-1 structure, is presented. The modulator is implemented with fully differential switched capacitor circuits in a standard 1 µm CMOS technology. The converter is powered by a single 5 V supply, uses two symmetrical reference voltages of 1 V, and is driven by a single 48 MHz clock. With an oversampling ratio of only 24 the converter achieves 91 dB of resolution, a peak SNR of 90 dB, and a peak SNDR of 85 dB at a Nyquist rate of 2 MHz after comb-filtering. The power consumption of the converter is 230 mW.Solid-State Circuits Conference, 1997. ESSCIRC '97. Proceedings of the 23rd European; 10/1997