Efforts Toward the Direct Experimental Characterization of Enzyme Microenvironments: Tyrosine100 in Dihydrofolate Reductase

State secrets : Site‐specific deuteration and FTIR studies reveal that Tyr100 in dihydrofolate reductase plays an important role in catalysis, with a strong electrostatic coupling occuring between Tyr100 and the charge that develops in the hydride‐transfer transition state (see picture, NADP⁺ purple, Tyr100 green). However, relaying correlated motions that facilitate catalysis from distal sites of the protein to the hydride donor may also be involved.

     

Expression of a Hybrid Human Superoxide Dismutase with a High Affinity for Heparin

IntroductionCopper,zinc- superoxide dismutases(SODs;EC.1.15 .1.1) are essential plasmic enzymes which cat-alyze the dismutation of highly reactive superoxideradical anions to hydrogen peroxide and oxygen so thatthe generation and clearance of oxygen free radicalsare regulated[1— 3 ] . The studies show that oxygen freeradicals play important roles in aging and the regula-tion of nitrogen monoxide[4,5] .The failure of the prop-er regulation will lead to inflammation,ischemicreperfusion injury…     

具有抗多种活性氧酶功能的金属卟啉的研究—钌卟啉仿酶活性测试

二价金属钌卟啉[Ru^Ⅱ(4-Cl-TPP)(CO)](1),[Ru^Ⅱ(4-MeO-TPP)(CO)](2),[Ru^Ⅱ(C,4,5-MeO-TPP)(CO)](3)和六价金属钌卟啉[Ru^Ⅵ（4Cl-TPP)(O)2](4),[Ru^Ⅵ(4-MeO-TPP)(O)2](5),[Ru^Ⅵ(3,4,5-MeO-TPP)(O)2](6)被合成,配合物1－6作为超氧化物歧化酶（SOD）和过氧化氢酶（CAT）功能模拟酶被测试,核黄素－蛋胺酸光照法证实,钌啉啉在不同浓度范围内有催化歧化O^.-2的作用,分光光度法证实能催化H2O2分解,用小鼠肝匀浆法测度试结果表明：金属钌啉具有良好的抗脂质过氧化作用,提出了金属钌卟啉在催化歧化超氧阴离子自由基（O^.-2)和催化分解过氧化氢（H2O2）协同过程中的可能机理,并讨论了卟啉环上不同取代基对催化活性的影响。     

Phen-铜(II)-氨基酸配合物的合成、表征及其SOD活性

合成了3个新的SOD模拟配合物：[Cu(Phen)(L-Gln)(H₂O)]Cl·2H₂O (1)、[Cu(Phen)(L-Ala)(H₂O)]Cl·4H₂O (2)、[Cu(Phen)(L-Thr)(H₂O)]Cl·2H₂O (3) [Phen(1，10-邻菲咯啉)、L-Gln(谷氨酰胺)、L-Ala(丙氨酸)、L-Thr(苏氨酸)]。用元素分析、摩尔电导、红外光谱、紫外-可见光谱对配合物进行了表征。用X-射线衍射对配合物[Cu(Phen)(L-Gln)(H₂O)]Cl·2H₂O的晶体结构进行了测定。用氯化硝基四氮唑蓝(NBT)光还原法对这3个配合物催化歧化超氧阴离子自由基(O₂^{- ·})的能力进行了测定。结果表明：这些配合物具有较高的SOD活性， 催化速率常数K_Q分别为1.58 × 10⁷ mol^-1·L·s^-1、5.65 × 10⁷ mol^-1·L·s^-1、0.83 × 10⁷ mol^-1·L·s^-1。     

四羧基锰酞菁作为超氧化物歧化酶和过氧化物酶模拟酶的研究

用核黄素 蛋氨酸光照法和黄嘌呤氧化酶 细胞色素C还原法证实,在1.0×10-6～1.0×10-5mol/L范围内四羧基锰酞菁(TcPcMn)表现出良好的清除超氧阴离子自由基(O—·2)的活性;用黄嘌呤氧化酶 NBT还原法测算出的TcPcMn清除O—·2的二级反应速率常数为7.77×105mol-1·L·s-1,表明TcPcMn作为超氧化物歧化酶(SOD)模拟酶能很好的抑制O—·2的还原性.TcPcMn既能催化H2O2与4 氨基安替比林和酚的显色反应,也能催化邻苯二胺的聚合,表明TcPcMn具有过氧化物酶(POD)活性.用邻苯三酚自氧法得出,TcPcMn将O—·2的氧化性转化成了POD和H2O2的氧化性.因此只要牺牲一定的POD底物,TcPcMn就可以清除掉因歧化O—·2而产生的H2O2,继而能避免因发生Fenton反应而产生氧化性高于O—·2的羟自由基,从而彻底消除O—·2的氧化性,与SOD相比,这是TcPcMn的一个优势.     

克氏原螯虾(Procambarus clarkii)应急二价铜离子时超氧化物歧化酶和过氧化氢酶的变化

用二价铜离子(Cu2+)胁迫克氏原螯虾,分析其肝胰腺、触角腺、鳃等组织的超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性变化.结果表明:克氏原螯虾能在含10mg/LCu2+水中生活;在10mg/LCu2+胁迫下,在0～24h,肝胰腺和触角腺中的CAT、SOD活性均显著升高,CAT活性在24h达到最高;在24～36h,肝胰腺和触角腺中的CAT活性开始下降,而SOD活性继续上升,在第36h时达到最高;48h后,肝胰腺和触角腺中SOD和SOD活性下降并均趋于平稳表达,SOD活性仍显著高于非胁迫处理时,而CAT活性则显著低于Cu2+处理时;鳃中的SOD和SOD活性未受到Cu2+浓度变化的影响.     

盐度对蛋白核小球藻生长、叶绿素荧光参数及代谢酶的影响

以蛋白核小球藻820为实验材料,研究了3种盐度(15、30、45)对其生长、叶绿素荧光参数和两种代谢酶活性的影响,以了解该小球藻对盐度的适应能力.结果表明,蛋白核小球藻820的生长随盐度增加而变慢;而油脂含量随盐度增加而升高.叶绿素荧光参数中的PSII最大光能转化效率(Fv/Fm)、PSII实际光能转化效率(ΦPSII)、光化学淬灭系数(qP)随盐度升高下降,而非光化学淬灭系数(NPQ)随盐度升高而上升.超氧化物歧化酶(SOD)活性变化大致趋势是低盐和高盐下活性较高,碳酸酐酶(CA)活性则随盐度升高而降低,第5 d时45盐度是30盐度培养的0.43倍.因此,认为高盐一定程度地抑制了蛋白核小球藻的生长、叶绿素荧光参数和CA活性,但是促进了总脂含量和抗氧化酶SOD活性的提高.     

Synergic approach to XAFS analysis for the identification of most probable binding motifs for mononuclear zinc sites in metalloproteins

In the present work a data analysis approach, based on XAFS data, is proposed for the identification of most probable binding motifs of unknown mononuclear zinc sites in metalloproteins. This approach combines multiple‐scattering EXAFS analysis performed within the rigid‐body refinement scheme, non‐muffin‐tin ab initio XANES simulations, average structural information on amino acids and metal binding clusters provided by the Protein Data Bank, and Debye–Waller factor calculations based on density functional theory. The efficiency of the method is tested by using three reference zinc proteins for which the local structure around the metal is already known from protein crystallography. To show the applicability of the present analysis to structures not deposited in the Protein Data Bank, the XAFS spectra of six mononuclear zinc binding sites present in diverse membrane proteins, for which we have previously proposed the coordinating amino acids by applying a similar approach, is also reported. By comparing the Zn K‐edge XAFS features exhibited by these proteins with those pertaining to the reference structures, key spectral characteristics, related to specific binding motifs, are observed. These case studies exemplify the combined data analysis proposed and further support its validity.