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Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding 总被引:3,自引:0,他引:3
We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes. 相似文献
994.
Grönberg KL Watmough NJ Thomson AJ Richardson DJ Field SJ 《The Journal of biological chemistry》2004,279(17):17120-17125
The bacterial respiratory nitric-oxide reductase (NOR) catalyzes the respiratory detoxification of nitric oxide in bacteria and Archaea. It is a member of the well known super-family of heme-copper oxidases but has a [heme Fe-non-heme Fe] active site rather than the [heme Fe-Cu(B)] active site normally associated with oxygen reduction. Paracoccus denitrificans NOR is spectrally characterized by a ligand-to-metal charge transfer absorption band at 595 nm, which arises from the high spin ferric heme iron of a micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site. On reduction of the nonheme iron, the micro-oxo bridge is broken, and the ferric heme iron is hydroxylated or hydrated, depending on the pH. At present, the catalytic cycle of NOR is a matter of much debate, and it is not known to which redox state(s) of the enzyme nitric oxide can bind. This study has used cyanide to probe the nature of the active site in a number of different redox states. Our observations suggest that the micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site represents a closed or resting state of NOR that can be opened by reduction of the non-heme iron. 相似文献
995.
A proteomic study of SUMO-2 target proteins 总被引:8,自引:0,他引:8
Vertegaal AC Ogg SC Jaffray E Rodriguez MS Hay RT Andersen JS Mann M Lamond AI 《The Journal of biological chemistry》2004,279(32):33791-33798
The SUMO family in vertebrates includes at least three distinct proteins (SUMO-1, -2, and -3) that are added as post-translational modifications to target proteins. A considerable number of SUMO-1 target proteins have been identified, but little is known about SUMO-2. A stable HeLa cell line expressing His6-tagged SUMO-2 was established and used to label and purify novel endogenous SUMO-2 target proteins. Tagged forms of SUMO-2 were functional and localized predominantly in the nucleus. His6-tagged SUMO-2 conjugates were affinity-purified from nuclear fractions and identified by mass spectrometry. Eight novel potential SUMO-2 target proteins were identified by at least two peptides. Three of these proteins, SART1, heterogeneous nuclear ribonucleoprotein (RNP) M, and the U5 small nuclear RNP 200-kDa helicase, play a role in RNA metabolism. SART1 and heterogeneous nuclear RNP M were both shown to be genuine SUMO targets, confirming the validity of the approach. 相似文献
996.
997.
Topoisomerase II is an essential enzyme that is targeted by a number of clinically valuable anticancer drugs. One class referred to as topoisomerase II poisons works by increasing the cellular level of topoisomerase II-mediated DNA breaks, resulting in apoptosis. Another class of topoisomerase II-directed drugs, the bis-dioxopiperazines, stabilizes the conformation of the enzyme where it attains an inactive salt-stable closed clamp structure. Bis-dioxopiperazines, similar to topoisomerase II poisons, induce cell killing, but the underlying mechanism is presently unclear. In this study, we use three different biochemically well characterized human topoisomerase IIalpha mutant enzymes to dissect the catalytic requirements needed for the enzyme to cause dominant sensitivity in yeast to the bis-dioxopirazine ICRF-193 and the topoisomerase II poison m-AMSA. We find that the clamp-closing activity, the DNA cleavage activity, and even both activities together are insufficient for topoisomerase II to cause dominant sensitivity to ICRF-193 in yeast. Rather, the strand passage event per se is an absolute requirement, most probably because this involves a simultaneous interaction of the enzyme with two DNA segments. Furthermore, we show that the ability of human topoisomerase IIalpha to cause dominant sensitivity to m-AMSA in yeast does not depend on clamp closure or strand passage but is directly related to the capability of the enzyme to respond to m-AMSA with increased DNA cleavage complex formation. 相似文献
998.
999.
Activation/deactivation of acetylcholinesterase by H2O2: more evidence for oxidative stress in vitiligo 总被引:5,自引:0,他引:5
Schallreuter KU Elwary SM Gibbons NC Rokos H Wood JM 《Biochemical and biophysical research communications》2004,315(2):502-508
Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3)M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6)M) activate this enzyme by increasing the Vmax>2-fold, meanwhile high concentrations of H2O2 (10(-3)M) inhibit the enzyme with a significant decrease in Vmax. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambdamax 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo. 相似文献
1000.