共查询到20条相似文献,搜索用时 15 毫秒
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Background
Bisubstrate enzymes, such as 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), exist in solution as an ensemble of conformations. 17β-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment.Methodology/Principal Findings
To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17β-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step.Conclusions/Significance
Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the βFαG′-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17β-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17β-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17β-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis. 相似文献3.
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In a new volume in the Handbooks of behavioural neuroscience series (series editor Joseph P. Huston), Randolf Menzel and Paul Benjamin have assembled an excellent and authoritative account of the importance, value and contribution of invertebrate models to the analysis of the molecular basis of learning and memory consolidation. 相似文献
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Amritraj Patra Leslie D. Nagy Qianqian Zhang Yan Su Livia Müller F. Peter Guengerich Martin Egli 《The Journal of biological chemistry》2014,289(24):16867-16882
DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion. 相似文献
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Arribas E Muñoz-Lopez A Garcia-Meseguer MJ Lopez-Najera A Avalos L Garcia-Molina F Garcia-Moreno M Varon R 《Bulletin of mathematical biology》2008,70(5):1425-1449
Taking as starting point the complete analysis of mean residence times in linear compartmental systems performed by Garcia-Meseguer
et al. (Bull. Math. Biol. 65:279–308, 2003) as well as the fact that enzyme systems, in which the interconversions between the different enzyme species involved are
of first or pseudofirst order, act as linear compartmental systems, we hereby carry out a complete analysis of the mean lifetime
that the enzyme molecules spend as part of the enzyme species, forms, or groups involved in an enzyme reaction mechanism.
The formulas to evaluate these times are given as a function of the individual rate constants and the initial concentrations
of the involved species at the onset of the reaction. We apply the results to unstable enzyme systems and support the results
by using a concrete example of such systems. The practicality of obtaining the mean times and their possible application in
a kinetic data analysis is discussed. 相似文献
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World Journal of Microbiology and Biotechnology - 相似文献
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Garima Srivastava Kritika Singh Mahe Talat Onkar Nath Srivastava Arvind M. Kayastha 《PloS one》2014,9(11)
β-Amylase finds application in food and pharmaceutical industries. Functionalized graphene sheets were customised as a matrix for covalent immobilization of Fenugreek β-amylase using glutaraldehyde as a cross-linker. The factors affecting the process were optimized using Response Surface Methodology based Box-Behnken design of experiment which resulted in 84% immobilization efficiency. Scanning and Transmission Electron Microscopy (SEM, TEM) and Fourier Tansform Infrared (FTIR) spectroscopy were employed for the purpose of characterization of attachment of enzyme on the graphene. The enzyme kinetic studies were carried out for obtaining best catalytic performance and enhanced reusability. Optimum temperature remained unchanged, whereas optimum pH showed shift towards acidic range for immobilized enzyme. Increase in thermal stability of immobilized enzyme and non-toxic nature of functionalized graphene can be exploited for production of maltose in food and pharmaceutical industries. 相似文献
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Kinetic changes of alpha-glucosidase from Saccharomyces cerevisiae in guanidinium chloride (GdmCl) and SDS solutions were investigated. The results showed both denaturants can lead conformational changes and loss of enzymatic activities. However, the concentrations of denaturants causing loss of activities were much lower than that of conformational changes, which suggested that the conformation of active site of α-glucosidase was more fragile than the whole molecular conformation in response to the two denaturants. According to the different kinetic process of the enzyme in the GdmCl and SDS solutions, the further investigation on the process of denaturation were made, it showed GdmCl and SDS had different types of inhibition and different types of interaction with the enzyme. Furthermore, the mechanisms of the two denaturants were discussed. 相似文献
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Infusini G Iannuzzi C Vilasi S Birolo L Pagnozzi D Pucci P Irace G Sirangelo I 《European biophysics journal : EBJ》2012,41(7):615-627
Myoglobin is an alpha-helical globular protein containing two highly conserved tryptophanyl residues at positions 7 and 14 in the N-terminal region. The simultaneous substitution of the two residues increases the susceptibility of the polypeptide chain to misfold, causing amyloid aggregation under physiological condition, i.e., neutral pH and room temperature. The role played by tryptophanyl residues in driving the folding process has been investigated by examining three mutated apomyoglobins, i.e., W7F, W14F, and the amyloid-forming mutant W7FW14F, by an integrated approach based on far-ultraviolet (UV) circular dichroism (CD) analysis, fluorescence spectroscopy, and complementary proteolysis. Particular attention has been devoted to examine the conformational and dynamic properties of the equilibrium intermediate formed at pH 4.0, since it represents the early organized structure from which the native fold originates. The results show that the W → F substitutions at position 7 and 14 differently affect the structural organization of the AGH subdomain of apomyoglobin. The combined effect of the two substitutions in the double mutant impairs the formation of native-like contacts and favors interchain interactions, leading to protein aggregation and amyloid formation. 相似文献