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Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases. 细菌、真菌和植物过氧化物酶超家族I类的系统发育关系。
Pub Date : 2004-08-01 DOI: 10.1111/j.1432-1033.2004.04262.x
Marcel Zámocký
Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.
分析了过氧化氢酶过氧化物酶、细胞色素c过氧化物酶和抗坏血酸过氧化物酶的分子系统发育。选择了60个代表性序列,涵盖了细菌、真菌和植物血红素过氧化物酶超家族I类的所有已知亚群。分析的每个序列都包含典型的过氧化物酶基序,这些基序经过进化可以有效地结合假体血红素群,从而实现过氧化物活性。对与祖先串联基因重复事件相匹配的过氧化氢酶-过氧化物酶的n端和c端结构域分别进行系统发育分析,以揭示其特定的进化历史。通过三种不同的方法获得的推断的无根系统发育树显示,在这个超家族的进化早期,存在四个明显分离的分支(过氧化氢酶过氧化物酶、抗坏血酸过氧化物酶和细胞色素c过氧化物酶的c端和n端结构域)。从结果来看,过氧化氢酶-过氧化物酶基因的重复事件发生在进化的后期,在这个阶段已经形成了所区分的过氧化物酶家族的个体特异性。有证据表明,血红素过氧化物酶超家族I类在原核生物和真核生物中广泛分布,遵循多基因家族进化的生灭过程。
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引用次数: 49
Relationships between structure, function and stability for pyridoxal 5'-phosphate-dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies. 可逆性辅因子解离研究揭示了愈伤棒状杆菌吡哆醛5'-磷酸依赖性淀粉磷酸化酶的结构、功能和稳定性之间的关系。
Pub Date : 2004-08-01 DOI: 10.1111/j.1432-1033.2004.04265.x
Richard Griessler, Barbara Psik, Alexandra Schwarz, Bernd Nidetzky

Using 0.4 m imidazole citrate buffer (pH 7.5) containing 0.1 mm l-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5'-phosphate and loss of activity. The inactivation rate of CcStP under resolution conditions at 30 degrees C was, respectively, four- and threefold reduced in two mutants, Arg234-->Ala and Arg242-->Ala, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem.270, 2126-2136]. The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5'-phosphate was increased up to 4.5-fold by added phosphate. The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state. Arg234-->Ala and Arg242-->Ala mutants showed, respectively, eight- and > 20-fold decreased apparent affinities for phosphate (K(app)), compared to the wild-type (K(app) approximately 6 mm). When reconstituted next to each other in solution, apo-protomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking. Pyridoxal-reconstituted CcStP was inactive but approximately 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mm) and phosphite (5 mm), respectively. pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pK(a) values for the cofactor 5'-phosphate and the exogenous oxyanion.

使用含有0.1 mm l-半胱氨酸的0.4 m咪唑柠檬酸缓冲液(pH 7.5), calluane棒状杆菌(CcStP)的同型二聚体淀粉磷酸化酶被解离成原生折叠亚基,同时释放5'-磷酸吡多醛并失去活性。在30℃的分解条件下,两个突变体Arg234- >Ala和Arg242- >Ala的失活率分别降低了四倍和三倍,这两个突变体先前被证明会导致CcStP的热稳定化[Griessler, R., Schwarz, A., Mucha, J.和Nidetzky, B.(2003)]。[j].中国生物医学工程学报,2016,33(2):444 - 444。用吡哆醛5′-磷酸重建野生型和突变型载脂蛋白磷酸化酶后,恢复的酶活性比例增加了4.5倍。对活性恢复的影响显示出对磷酸盐浓度的饱和依赖,并且是与氧离子相互作用的结果,这是特定于第四态的。与野生型(K(app)约6 mm)相比,Arg234- >Ala和Arg242- >Ala突变体对磷酸盐(K(app))的表观亲和力分别降低了8倍和20倍以上。当在溶液中相邻重组时,CcStP和大肠杆菌麦芽糊精磷酸化酶的载脂蛋白原体没有检测到与杂交二聚体的关联,表明不同亚基之间缺乏结构互补性。吡多醛重组的CcStP是无活性的,但在pH为7.5时,磷酸盐(3 mm)和亚磷酸盐(5 mm)分别可以恢复大约60%和5%的野生型活性。pH对天然酶和与磷酸盐结合的吡哆醛磷酸化酶的催化速率的影响不同,这可以反映辅因子5'-磷酸和外源氧阴离子的pK(a)值的差异。
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引用次数: 10
Akt-dependent phosphorylation negatively regulates the transcriptional activity of dHAND by inhibiting the DNA binding activity. akt依赖性磷酸化通过抑制DNA结合活性负向调节dHAND的转录活性。
Pub Date : 2004-08-01 DOI: 10.1111/j.1432-1033.2004.04267.x
Masao Murakami, Keiichiro Kataoka, Shigetomo Fukuhara, Osamu Nakagawa, Hiroki Kurihara

HAND2/dHAND is a basic helix-loop-helix transcription factor expressed in the heart and neural crest derivatives during embryogenesis. Although dHAND is essential for branchial arch, cardiovascular and limb development, its target genes have not been identified. The regulatory mechanisms of dHAND function also remain relatively unknown. Here we report that Akt/PKB, a serine/threonine protein kinase involved in cell survival, growth and differentiation, phosphorylates dHAND and inhibits dHAND-mediated transcription. AU5-dHAND expressed in 293T cells became phosphorylated, possibly at its Akt phosphorylation motif, in the absence of kinase inhibitors, whereas the phosphatidylinositol 3-kinase inhibitor wortmannin and the Akt inhibitor NL-71-101, but not the p70 S6 kinase inhibitor rapamycin, significantly reduced dHAND phosphorylation. Coexpression of HA-Akt augmented dHAND phosphorylation at multiple serine and threonine residues mainly located in the bHLH domain and, as a result, decreased the transcriptional activity of dHAND. Consistently, alanine mutation mimicking the nonphosphorylation state abolished the inhibitory effect of Akt on dHAND, whereas aspartate mutation mimicking the phosphorylation state resulted in a loss of dHAND transcriptional activity. These changes in dHAND transcriptional activity were in parallel with changes in the DNA binding activity rather than in dimerization activity. These results suggest that Akt-mediated signaling may regulate dHAND transcriptional activity through the modulation of its DNA binding activity during embryogenesis.

HAND2/dHAND是一种基本的螺旋-环-螺旋转录因子,在胚胎发生过程中在心脏和神经嵴衍生物中表达。虽然dHAND对鳃弓、心血管和肢体发育至关重要,但其靶基因尚未确定。dHAND功能的调控机制也相对未知。Akt/PKB是一种丝氨酸/苏氨酸蛋白激酶,参与细胞存活、生长和分化,磷酸化dHAND并抑制dHAND介导的转录。在缺乏激酶抑制剂的情况下,293T细胞中表达的AU5-dHAND被磷酸化,可能是在其Akt磷酸化基元上,而磷脂酰肌醇3-激酶抑制剂wortmannin和Akt抑制剂NL-71-101,而p70 S6激酶抑制剂rapamycin则没有显著降低dHAND的磷酸化。HA-Akt的共表达增强了dHAND主要位于bHLH结构域的多个丝氨酸和苏氨酸残基的磷酸化,从而降低了dHAND的转录活性。同样,模拟非磷酸化状态的丙氨酸突变消除了Akt对dHAND的抑制作用,而模拟磷酸化状态的天冬氨酸突变导致dHAND转录活性丧失。这些dHAND转录活性的变化与DNA结合活性的变化而不是与二聚化活性的变化平行。这些结果表明,在胚胎发生过程中,akt介导的信号可能通过调节dHAND的DNA结合活性来调节dHAND的转录活性。
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引用次数: 18
Substrate specificity and transport mode of the proton-dependent amino acid transporter mPAT2. 质子依赖性氨基酸转运体mPAT2的底物特异性和转运方式。
Pub Date : 2004-08-01 DOI: 10.1111/j.1432-1033.2004.04268.x
Martin Foltz, Carmen Oechsler, Michael Boll, Gabor Kottra, Hannelore Daniel

The PAT2 transporter has been shown to act as an electrogenic proton/amino acid symporter. The PAT2 cDNA has been cloned from various human, mouse and rat tissues and belongs to a group of four genes (pat1 to pat4) with PAT3 and PAT4 still resembling orphan transporters. The first immunolocalization studies demonstrated that the PAT2 protein is found in the murine central nervous system in neuronal cells with a proposed role in the intra and/or intercellular amino acid transport. Here we provide a detailed analysis of the transport mode and substrate specificity of the murine PAT2 transporter after expression in Xenopus laevis oocytes, by electrophysiological techniques and flux studies. The structural requirements to the PAT2 substrates - when considering both low and high affinity type substrates - are similar to those reported for the PAT1 protein with the essential features of a free carboxy group and a small side chain. For high affinity binding, however, PAT2 requires the amino group to be located in an alpha-position, tolerates only one methyl function attached to the amino group and is highly selective for the L-enantiomers. Electrophysiological analysis revealed pronounced effects of membrane potential on proton binding affinity, but substrate affinities and maximal transport currents only modestly respond to changes in membrane voltage. Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.

PAT2转运体已被证明是一种电致质子/氨基酸同向转运体。PAT2 cDNA已从各种人类、小鼠和大鼠组织中克隆出来,属于一组四个基因(pat1至pat4),其中PAT3和pat4仍然类似于孤儿转运蛋白。首次免疫定位研究表明,在小鼠中枢神经系统的神经元细胞中发现了PAT2蛋白,并提出了在细胞内和/或细胞间氨基酸运输中的作用。在这里,我们通过电生理技术和通量研究,详细分析了小鼠PAT2转运体在非洲爪蟾卵母细胞中表达后的转运方式和底物特异性。对PAT2底物的结构要求-当考虑低亲和力和高亲和力类型底物时-与报道的PAT1蛋白相似,具有游离羧基和小侧链的基本特征。然而,对于高亲和力结合,PAT2要求氨基位于α位置,仅耐受与氨基连接的一个甲基功能,并且对l -对映体具有高度选择性。电生理分析表明,膜电位对质子结合亲和性有显著影响,但底物亲和性和最大输运电流对膜电压的变化响应不大。底物亲和力依赖于细胞外pH值,而质子与PAT2的结合亲和力与底物无关,有利于质子与底物的顺序结合。最大输运电流依赖于衬底,这表明负载载流子向内部的移位是速率限制步骤。
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引用次数: 34
Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms. 血小板因子4通过KDR依赖和独立机制破坏血管内皮生长因子诱导的细胞内信号级联。
Pub Date : 2004-08-01 DOI: 10.1111/j.1432-1033.2004.04263.x
Eric Sulpice, Jean-Olivier Contreres, Julie Lacour, Marijke Bryckaert, Gerard Tobelem

The mechanism by which the CXC chemokine platelet factor 4 (PF-4) inhibits endothelial cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent vascular endothelial growth factor 165 (VEGF(165)) and fibroblast growth factor 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF(165), which binds heparin, and by VEGF(121), which does not. Activation of the VEGF receptor, KDR, and phospholipase Cgamma (PLCgamma) was unaffected in conditions in which PF-4 inhibited VEGF(121)-induced DNA synthesis. In contrast, VEGF(165)-induced phosphorylation of KDR and PLCgamma was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF(165), but not that of VEGF(121), to KDR. PF-4 also strongly inhibited the VEGF(165)- and VEGF(121)-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF(165) it interacts directly or upstream from Raf1; for VEGF(121), it acts downstream from PLCgamma. Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF(121) and VEGF(165), involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.

CXC趋化因子血小板因子4 (PF-4)抑制内皮细胞增殖的机制尚不清楚。据报道,PF-4的肝素结合结构域可阻止血管内皮生长因子165 (VEGF(165))和成纤维细胞生长因子2 (FGF2)与其受体相互作用。然而,其他研究表明,PF-4通过肝素结合的独立相互作用起作用。在这里,我们比较了PF-4对与肝素结合的VEGF(165)和不与肝素结合的VEGF(121)诱导的增殖相关的信号事件的影响。在PF-4抑制VEGF(121)诱导的DNA合成的条件下,VEGF受体、KDR和磷脂酶Cgamma (PLCgamma)的激活不受影响。相反,VEGF(165)诱导的KDR和PLCgamma磷酸化被PF-4部分抑制。这些观察结果与PF-4影响VEGF与KDR的结合一致(165),但不影响VEGF与KDR的结合(121)。PF-4还强烈抑制VEGF(165)-和VEGF(121)诱导的包括Raf1、MEK1/2和ERK1/2在内的丝裂原活化蛋白(MAP)激酶信号通路:对于VEGF(165),它直接或上游作用于Raf1;对于VEGF(121),它作用于PLCgamma的下游。最后,PF-4抑制由VEGF(121)和VEGF(165)诱导的内皮细胞增殖的机制,涉及KDR下游MAP激酶信号通路的破坏,似乎不涉及CXCR3B的激活。
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引用次数: 31
A new framework for the estimation of control parameters in metabolic pathways using lin-log kinetics. 利用林对数动力学估计代谢途径控制参数的新框架。
Pub Date : 2004-08-01 DOI: 10.1111/j.0014-2956.2004.04269.x
Liang Wu, Weiming Wang, Wouter A van Winden, Walter M van Gulik, Joseph J Heijnen

The control properties of biochemical pathways can be described by control coefficients and elasticities, as defined in the framework of metabolic control analysis. The determination of these parameters using the traditional metabolic control analysis relationships is, however, limited by experimental difficulties (e.g. realizing and measuring small changes in biological systems) and lack of appropriate mathematical procedures (e.g. when the more practical large changes are made). In this paper, the recently developed lin-log approach is proposed to avoid the above-mentioned problems and is applied to estimate control parameters from measurements obtained in steady state experiments. The lin-log approach employs approximative linear-logarithmic kinetics parameterized by elasticities and provides analytical solutions for fluxes and metabolite concentrations when large changes are made. Published flux and metabolite concentration data are used, obtained from a reconstructed section of glycolysis converting 3-phosphoglycerate to pyruvate [Giersch, C. (1995) Eur. J. Biochem. 227, 194-201]. With the lin-log approach, all data from different experiments can be combined to give realistic elasticity and flux control coefficient estimates by linear regression. Despite the large changes, a good agreement of fluxes and metabolite concentrations is obtained between the measured and calculated values according to the lin-log model. Furthermore, it is shown that the lin-log approach allows a rigorous statistical evaluation to identify the optimal reference state and the optimal model structure assumption. In conclusion, the lin-log approach addresses practical problems encountered in the traditional metabolic control analysis-based methods by introducing suitable nonlinear kinetics, thus providing a novel framework with improved procedures for the estimation of elasticities and control parameters from large perturbation experiments.

生化途径的控制特性可以用控制系数和弹性来描述,在代谢控制分析的框架中定义。然而,使用传统的代谢控制分析关系来确定这些参数受到实验困难(例如,实现和测量生物系统中的微小变化)和缺乏适当的数学程序(例如,当进行更实际的大变化时)的限制。为了避免上述问题,本文提出了最近发展起来的林对数方法,并将其应用于从稳态实验中得到的测量值估计控制参数。林-对数方法采用近似的线性-对数动力学参数化的弹性,并提供分析解决方案,当通量和代谢物浓度的大变化。已发表的通量和代谢物浓度数据,从糖酵解将3-磷酸甘油酸转化为丙酮酸的重构部分获得[Giersch, C. (1995) Eur。[j].生物化学学报,2016,33(2):444 - 444。使用林对数方法,可以将不同实验的所有数据结合起来,通过线性回归给出真实的弹性和通量控制系数估计。尽管变化很大,但根据林-对数模型,在通量和代谢物浓度的测量值和计算值之间获得了很好的一致性。此外,lin-log方法可以通过严格的统计评估来确定最优参考状态和最优模型结构假设。总之,林对数方法通过引入适当的非线性动力学,解决了传统基于代谢控制分析方法中遇到的实际问题,从而为大扰动实验中弹性和控制参数的估计提供了一个新的框架和改进的程序。
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引用次数: 81
Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold-adaptation. 来自嗜冷细菌希瓦氏杆菌SIB1的FKBP家族成员蛋白可能参与冷适应。
Pub Date : 2004-04-01 DOI: 10.1111/j.1432-1033.2004.04049.x
Yutaka Suzuki, Mitsuru Haruki, Kazufumi Takano, Masaaki Morikawa, Shigenori Kanaya

A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 degrees C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 degrees C compared to that at 20 degrees C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 degrees C with a k(cat)/K(m) value of 0.87 micro m(-1) x s(-1). When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T(1) refolding assay at 10 and 20 degrees C, the protein exhibited higher activity at 10 degrees C with a k(cat)/K(m) value of 0.50 micro m(-1) x s(-1). These k(cat)/K(m) values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria.

在4℃和20℃条件下培养一株希瓦氏菌SIB1,用二维聚丙烯酰胺凝胶电泳法对提取的总可溶性蛋白进行分析。这些模式的对比分析结果显示,细胞蛋白质含量与28 kDa的分子量和等电点四个在4摄氏度而大大增加,在20度C测定的n端氨基酸序列,其次是克隆和测序的基因编码的蛋白质,表明这种蛋白质是FKBP家族的一员的蛋白质氨基酸序列的身份56%,大肠杆菌FKBP22。该蛋白以his标记的形式在大肠杆菌中过量生产,纯化并分析肽基脯氨酸顺式反式异构酶活性。以n -琥珀酰- ala - leu - pro - ph -p-硝基苯胺为底物,在不同温度下通过蛋白酶偶联试验测定其活性,该蛋白在10℃时活性最高,k(cat)/ k(m)值为0.87 μ m(-1) × s(-1)。用RNase T(1)重折叠法测定肽基脯氨酸顺式反式异构酶在10℃和20℃下的活性时,该蛋白在10℃下表现出更高的活性,k(cat)/ k(m)值为0.50 μ m(-1) x s(-1)。这些k(cat)/ k(m)值较低,但与大肠杆菌FKBP22相当。我们认为FKBP家族蛋白参与了嗜冷细菌的冷适应。
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引用次数: 66
Disruption of the interaction between the Rieske iron-sulfur protein and cytochrome b in the yeast bc1 complex owing to a human disease-associated mutation within cytochrome b. 酵母bc1复合体中Rieske铁硫蛋白与细胞色素b的相互作用因细胞色素b的人类疾病相关突变而中断
Pub Date : 2004-04-01 DOI: 10.1111/j.1432-1033.2004.04036.x
Nicholas Fisher, Ingrid Bourges, Philip Hill, Gael Brasseur, Brigitte Meunier

The mitochondrial cytochrome b missense mutation, G167E, has been reported in a patient with cardiomyopathy. The residue G167 is located in an extramembranous helix close to the hinge region of the iron-sulfur protein. In order to characterize the effects of the mutation on the structure and function of the bc(1) complex, we introduced G167E into the highly similar yeast cytochrome b. The mutation had a severe effect on the respiratory function, with the activity of the bc(1) complex decreased to a few per cent of the wild type. Analysis of the enzyme activity indicated that the mutation affected its stability, which could be the result of an altered binding of the iron-sulfur protein on the complex. G167E had no major effect on the interaction between the iron-sulfur protein headgroup and the quinol oxidation site, as judged by the electron paramagnetic resonance signal, and only a minor effect on the rate of cytochrome b reduction, but it severely reduced the rate of cytochrome c(1) reduction. This suggested that the mutation G167E could hinder the movement of the iron-sulfur protein, probably by distorting the structure of the hinge region. The function of bc(1) was partially restored by mutations (W164L and W166L) located close to the primary change, which reduced the steric hindrance caused by G167E. Taken together, these observations suggest that the protein-protein interaction between the n-sulfur protein hinge region and the cytochrome b extramembranous cd2 helix is important for maintaining the structure of the hinge region and, by consequence, the movement of the headgroup and the integrity of the enzyme.

线粒体细胞色素b错义突变,G167E,已报道在心肌病患者。残基G167位于靠近铁硫蛋白铰链区域的膜外螺旋上。为了表征突变对bc(1)复合体结构和功能的影响,我们将G167E引入高度相似的酵母细胞色素b中。突变对呼吸功能有严重影响,bc(1)复合体的活性下降到野生型的百分之几。酶活性分析表明,突变影响了其稳定性,这可能是铁硫蛋白在复合物上的结合改变的结果。从电子顺磁共振信号判断,G167E对铁硫蛋白头基团与喹啉氧化位点的相互作用无明显影响,对细胞色素b的还原速率影响较小,但严重降低了细胞色素c(1)的还原速率。这表明突变G167E可能通过扭曲铰链区域的结构来阻碍铁硫蛋白的运动。位于原突变位点附近的突变(W164L和W166L)部分恢复了bc(1)的功能,减少了G167E引起的位阻。综上所述,这些观察结果表明,n-硫蛋白铰链区域和细胞色素b膜外cd2螺旋之间的蛋白质-蛋白质相互作用对于维持铰链区域的结构,从而维持头基团的运动和酶的完整性是重要的。
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引用次数: 40
Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate. 高浓度底物对黑腹果蝇乙酰胆碱酯酶的抑制作用。
Pub Date : 2004-04-01 DOI: 10.1111/j.1432-1033.2004.04048.x
Jure Stojan, Laure Brochier, Carole Alies, Jacques Philippe Colletier, Didier Fournier

Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.

在高底物浓度下,乙酰胆碱酯酶对乙酰胆碱的水解受到抑制。为了确定参与这一现象的残基,我们对活性位点峡谷的大部分残基进行了突变,但这些突变并没有完全消除水解。因此,我们分析了不可水解的底物类似物对底物水解和乙酰酶类似物再激活的影响。对各种模型的分析使我们提出了以下事件序列:底物最初在活性位点峡谷边缘结合,然后滑到峡谷底部水解。另一种底物分子可以与外周位点结合:(a)当胆碱仍在峡谷内时,它将因此阻碍其退出;(b)在胆碱解离后,但在去乙酰化发生之前,外周位点的结合增加了去乙酰化速率,但(c)如果在催化丝氨酸去乙酰化之前,与外周位点结合的底物分子滑到活性位点的底部,它的新位置将阻止水的接近,从而阻断去乙酰化。
{"title":"Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate.","authors":"Jure Stojan,&nbsp;Laure Brochier,&nbsp;Carole Alies,&nbsp;Jacques Philippe Colletier,&nbsp;Didier Fournier","doi":"10.1111/j.1432-1033.2004.04048.x","DOIUrl":"https://doi.org/10.1111/j.1432-1033.2004.04048.x","url":null,"abstract":"<p><p>Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.</p>","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":"271 7","pages":"1364-71"},"PeriodicalIF":0.0,"publicationDate":"2004-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04048.x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40849985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 42
Protein engineering of pyruvate carboxylase: investigation on the function of acetyl-CoA and the quaternary structure. 丙酮酸羧化酶的蛋白质工程:乙酰辅酶a的功能及其四级结构的研究。
Pub Date : 2004-04-01 DOI: 10.1111/j.1432-1033.2004.04051.x
Shinji Sueda, Md Nurul Islam, Hiroki Kondo

Pyruvate carboxylase (PC) from Bacillus thermodenitrificans was engineered in such a way that the polypeptide chain was divided into two, between the biotin carboxylase (BC) and carboxyl transferase (CT) domains. The two proteins thus formed, PC-(BC) and PC-(CT+BCCP), retained their catalytic activity as assayed by biotin-dependent ATPase and oxamate-dependent oxalacetate decarboxylation, for the former and the latter, respectively. Neither activity was dependent on acetyl-CoA, in sharp contrast to the complete reaction of intact PC. When assessed by gel filtration chromatography, PC-(BC) was found to exist either in dimers or monomers, depending on the protein concentration, while PC-(CT + BCCP) occurred in dimers for the most part. The two proteins do not associate spontaneously or in the presence of acetyl-CoA. Based on these observations, this paper discusses how the tetrameric structure of PC is built up and how acetyl-CoA modulates the protein structure.

对热反硝化芽孢杆菌的丙酮酸羧化酶(PC)进行了工程设计,使其多肽链在生物素羧化酶(BC)和羧基转移酶(CT)结构域之间分为两个结构域。由此形成的两种蛋白PC-(BC)和PC-(CT+BCCP)分别通过生物素依赖的atp酶和草酸依赖的草酸脱羧检测保持了它们的催化活性。这两种活性都不依赖于乙酰辅酶a,与完整PC的完全反应形成鲜明对比。通过凝胶过滤层析分析,发现PC-(BC)存在于二聚体或单体中,这取决于蛋白质浓度,而PC-(CT + BCCP)大部分存在于二聚体中。这两种蛋白质不能自发结合或在乙酰辅酶a存在下结合。在此基础上,本文讨论了PC的四聚体结构是如何建立的,以及乙酰辅酶a是如何调节蛋白质结构的。
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引用次数: 25
期刊
European journal of biochemistry
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