J. Appl. Cryst.の発刊に際して

基礎講座 電気泳動(Electrophoresis)

Advances in CO2 capture technology—The U.S. Department of Energy's Carbon Sequestration Program ☆

The first suggestion that physical exercise results in free radical-mediated damage to tissues appeared in 1978, and the past three decades have resulted in a large growth of knowledge regarding exercise and oxidative stress. Although the sources of oxidant production during exercise continue to be debated, it is now well established that both resting and contracting skeletal muscles produce reactive oxygen species and reactive nitrogen species. Importantly, intense and prolonged exercise can result in oxidative damage to both proteins and lipids in the contracting myocytes. Furthermore, oxidants can modulate a number of cell signaling pathways and regulate the expression of multiple genes in eukaryotic cells. This oxidant-mediated change in gene expression involves changes at transcriptional, mRNA stability, and signal transduction levels. Furthermore, numerous products associated with oxidant-modulated genes have been identified and include antioxidant enzymes, stress proteins, DNA repair proteins, and mitochondrial electron transport proteins. Interestingly, low and physiological levels of reactive oxygen species are required for normal force production in skeletal muscle, but high levels of reactive oxygen species promote contractile dysfunction resulting in muscle weakness and fatigue. Ongoing research continues to probe the mechanisms by which oxidants influence skeletal muscle contractile properties and to explore interventions capable of protecting muscle from oxidant-mediated dysfunction.

/pdf/exercise-induced-oxidative-stress-cellular-mechanisms-and-4a18810890.pdf

Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production

Excessive reactive oxygen species Revised abstract, especially superoxide anion (O2•−), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis Superoxide dismutases (SODs) are the major antioxidant defense systems against O2•−, which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of O2•− H2O2, which may participate in cell signaling In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer Given the essential role

Superoxide dismutases: role in redox signaling, vascular function, and diseases.

To test the hypothesis that histidine 64 in the active site of human carbonic anhydrase II functions as a proton-transfer group in the catalysis of CO2 hydration, we have studied a site-specific mutant having histidine 64 replaced by alanine, which cannot transfer protons. The steady-state kinetics of CO2 hydration has been measured as well as the exchange of 18O between CO2 and water at chemical equilibrium. The results show that the rate of exchange between CO2 and HCO3- at chemical equilibrium is essentially unaffected by the amino acid substitution at pH greater than 7.0 and slightly decreased in the mutant at pH less than 7.0 (by a factor of 2 at pH 6.0). However, in the absence of buffer the rate of release from the active site of water bearing substrate oxygen is smaller by as much as 20-fold for the mutant as compared to unmodified enzyme. Furthermore, in the unmodified enzyme water release is inhibited by micromolar concentrations of Cu2+ ions, but no such inhibition is observed with the alanine 64 variant. These results suggest that the mutation has specifically affected the rate of proton transfer between the active site and the reaction medium. This kinetic defect in the mutant can be overcome by increasing the concentration of certain buffers, such as imidazole and 1-methylimidazole, but not by others buffers, such as MOPS or HEPES. Similarly, the maximal rate of CO2 hydration at steady state catalyzed by the alanine 64 variant is very low in the presence of MOPS or TAPS buffers but considerably higher in the presence of imidazole derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant

Human carbonic anhydrase II (HCA II) is a zinc-metalloenzyme that catalyzes the reversible interconversion of CO2 and HCO3-. The rate-limiting step of this catalysis is the transfer of a proton between the Zn-bound solvent molecule and residue His64. In order to fully characterize the active site structural features implicated in the proton transfer mechanism, the refined X-ray crystal structure of uncomplexed wild type HCA II to 1.05 A resolution with an Rcryst value of 12.0% and an Rfree value of 15.1% has been elucidated. This structure provides strong clues as to the pathway of the intramolecular proton transfer between the Zn-bound solvent and His64. The structure emphasizes the role of the solvent network, the unique positioning of solvent molecule W2, and the significance of the dual conformation of His64 in the active site. The structure is compared with molecular dynamics (MD) simulation calculations of the Zn-bound hydroxyl/His64+ (charged) and the Zn-bound water/His64 (uncharged) HCA II states....

Atomic crystal and molecular dynamics simulation structures of human carbonic anhydrase II: insights into the proton transfer mechanism

In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants:  H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1−10.0, and the catalysis of the exchange of 18O between CO2 and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 105 s-1 was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 103 s-1 was observed for His62. The observation, in the crystal structures, of a completed ...

Chingkuang Tu

Papers

Role of histidine 64 in the catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant

Atomic crystal and molecular dynamics simulation structures of human carbonic anhydrase II: insights into the proton transfer mechanism

Structural and Kinetic Characterization of Active-Site Histidine as a Proton Shuttle in Catalysis by Human Carbonic Anhydrase II

Catalytic Properties of Human Manganese Superoxide Dismutase

Rate of exchange of water from the active site of human carbonic anhydrase C