CYP 119过氧化物酶及其215位突变体在苯乙烯类底物环氧化反应中的作用

通过对CYP119过氧化物酶活性中心215位保守苏氨酸的定点突变,研究突变体对催化苯乙烯类似底物环氧化反应的作用效率。考察了CYP119酶215位突变体的热稳定性;端基双键的苯乙烯类似物和非端基双键的β-甲基苯乙烯类似物环氧化反应的转化率和对映选择性。结果表明,CYP119酶突变体T215G和T215V催化端基双键类底物效果与亲本酶类似,而催化非端基双键类底物在转化率和对映选择性上都具有一定变化。突变体T215G催化苯乙烯环氧化反应的转化率高达99.6%。突变体T215G能一定程度提高催化顺式β-甲基苯乙烯环氧化反应的对映选择性和转化率。突变体T215V催化顺式-β-甲基苯乙烯类底物,环氧化反应对映选择性远低于亲本酶和突变体T215G。说明215位苏氨酸的氢键效应和侧链氨基酸大小对CYP119酶催化特定底物的环氧化反应具有一定影响。     

共生真菌巴西类壳小圆孢中环二肽类代谢产物研究

利用正相和反相柱色谱,Sephadex LH-20凝胶柱色谱及反相HPLC等方法从昆虫共生真菌Paraconiothyrium brasiliense的发酵产物中分离得到9个环二肽类化合物,经NMR,MS及理化性质等方法,结构确定为环(L-亮氨酸-L-酪氨酸)(1),环(L-色氨酸-L-脯氨酸)(2),环(L-脯氨酸-L-苯丙氨酸)(3),环(L-脯氨酸-L-酪氨酸)(4),环(L-脯氨酸-L-亮氨酸)(5),环(L-亮氨酸-反式-4-羟基-L-脯氨酸)(6),环(L-异亮氨酸-L-脯氨酸)(7),环(L-缬氨酸-L-脯氨酸)(8),环(L-亮氨酸-L-异亮氨酸)(9)。所有化合物均为首次从该属菌种中分离得到。     

pH对增强生物除磷系统酶活性的影响

通过比较不同pH值下增强生物除磷系统中关键酶活性的变化规律, 研究了酶活性与聚磷菌污泥产率系数及可溶性正磷酸盐(SOP)的关系. 结果表明, 在pH=6.4-7.6范围内, 脱氢酶、腺苷酸激酶和聚磷酸盐激酶的活性随着pH的增加而线性增加, 酸性磷酸酶和碱性磷酸酶的活性不受pH的影响. 聚磷菌的产率系数与脱氢酶活性、厌氧释磷速率与腺苷酸激酶活性、好氧吸磷速率与聚磷酸盐激酶活性分别呈线性关系. 表明较高的pH有利于聚磷菌的生长和提高聚磷菌的活性, 从而提高了除磷效率.     

2′,5′-寡核苷酸C_(2′P_5′)C_(2′P_5′)A的合成

近年来发现用干扰素处理过的细胞胞浆提取物和双链RNA、ATP一起培养,可以生成一个低分子量的蛋白合成抑制剂,即5′-三磷酸-三聚2′,5′-腺苷酸(Ⅰ)。由于(Ⅰ)具有天然核酸中不存在的2′,5′-磷酸酯链以及它的抗病毒活性,因此引起不少化学家的兴趣,已有不少文章报导了(Ⅰ)及其类似物的合成,同时也发现不含三磷酸的三聚2′,5′-腺苷酸(Ⅱ)也同样具有活性。为了进一步研究(Ⅰ)的结构与活性关系,本文报导了用胞嘧啶碱基代替腺嘌呤碱基的(Ⅱ)的类似物C_(2′P5′)C_(2′P5′)A(Ⅲ)的合成。     

三磷酸腺苷大量生产方法研究成功

三磷酸腺苷(ATP)是许多酶催化反应所需要的供应能量的辅助因子(Cofactor)。最近,美国麻省理工学院G.M.Whitesides博士领导的研究组宣布他们研究成功了一种能大量生产ATP的方法,即将丙酮热解生成乙烯酮,再与磷酸进行加成而得乙酰磷酸,后者在腺苷激酶、腺苷酸激酶和乙酸激酶等三种酶共同作用下将腺苷进行磷酸化而成一磷酸腺苷(AMP)、二磷酸     

基于[3+2]环加成反应的氮杂螺[3,4]环辛烷的合成

哌嗪、吗啉是小分子药物中常见的结构片段,2,6-二氮杂螺[3,4]环辛烷和2-氧-6-氮杂螺[3,4]环辛烷等四元螺环化合物作为其类似物,在药物化学领域具有良好的应用前景.本研究以廉价易得的丙二酸二乙酯或丙烯酸乙酯为起始原料,经[3+2]环加成反应,可便捷高效地完成6-苄基-2,6-二氮杂螺[3,4]环辛烷和2-氧-6-氮杂螺[3,4]环辛烷克级水平的合成.     

烟酰胺腺嘌呤二核苷酸(NAD~+)类似物的合成及与NAD~+依赖型酶相互作用进展

烟酰胺腺嘌呤二核苷酸(NAD+)与NAD+依赖型酶间的相互作用在细胞氧化还原反应、能量代谢、信号转导过程中发挥着重要作用.NAD+依赖型酶利用共同的辅酶,它们之间相互作用规律受到越来越广泛的关注.主要针对NAD+类似物的合成及与NAD+依赖型酶相互作用研究的最新进展作一综述,包括NAD+类似物的合成及其与天然NAD+依赖型酶相互作用、NAD+与突变酶相互作用及NAD+类似物与突变酶相互作用.     

活性维生素D3类似物的合成及构效关系研究

1α,25-二羟基维生素D₃(1α,25-(OH)₂-D₃,125D)是维生素D中最具生理活性的代谢产物,但因高钙血症副反应而限制其临床应用。从对构效关系的研究出发,迄今已合成三千多种类似物。本文综述了近年来对某些A环修饰(C2位修饰、C3位修饰、芳香A环类似物、A环开环类似物)、侧链修饰、CD环修饰、seco-B环修饰和非开环甾体的活性维生素 D₃ 类似物的设计、合成以及构效关系的研究,旨在为新型较佳活性维生素D₃类似物的合成及临床开发提供参考。     

质体醌类化合物的合成研究

醌环的取代基对醌类化合物进行光合作用的电子传递过程有很大影响. 我们设想若能在保持与醌环上的甲基相近体积的条件下, 将其中一个甲基换成氯原子, 这样, 这类化合物醌环上的电子密度显然会低于质体醌, 从而进一步研究它们在光合作用中的作用, 有助于进一步阐明光合作用机制, 以3-氯-2-甲基苯胺为原料, 经氧化, 自由烷基化反应, 合成了十六个具有不同长侧链结构的质体醌类似物.     

五氯代-12H-双苯并[1, 3, 2]二氧磷杂八环的合成与化学

以双(2-羟基-3,5-二氯苯基)甲烷(4)与PSCl3关环,高收率地得到2,4,6,8,10-五氯-6-硫-12H-双苯并[d,g][1,3,2]二氧磷杂八环(5)。5与酚在无水K2CO3及铜粉存在下,或与醇在醇钠存在下反应,生成2,4,8,10-四氯-6-硫-6-芳氧基-12H-双苯并[1,3,2]二氧磷杂八环(6),或6-烷氧基的类似物(7)。5与醇在三乙胺存在下反应的产物为2,4,8,10-四氯-6-硫-6-羟基-12H-双苯并[1,3,2]二氧磷杂八环三乙胺盐(8)。8在DMSO中回流则氧化为它的氧类似物9。     

氨基酸衍生物的反应、合成和生物活性研究

讨论了我们小组近年来以氨基酸衍生物为主线，在代谢型谷氨酸受体及蛋白激酶C有选择性的调节剂的发现和合成，在发展新反应以及天然产物合成方面所取得的一些结果。     

Bile acid acyl adenylate: a possible intermediate to produce a protein-bound bile acid

The non-enzymatic production of a protein-bound adduct by the action of the acyl adenylate of bile acids is described. On incubation of deoxycholyl adenylate with substance P in phosphate buffer, peptides covalently bound with one or two molecules of the bile acid were detected. The modified peptides were structurally characterized by time-of-flight mass spectrometry with matrix-assisted laser desorption/ionization (MALDI-TOFMS) in the post-source decay mode, and by liquid chromatography/electrospray ionization MS/MS. The deoxycholic acid was bound on substance P through the amino group at Arg-1 and/or Lys-3. The adenylate of cholic acid also produced the protein-bound bile acid on incubation with lysozyme, and the binding sites of the cholic acid appeared to be the lysine residues at 1, 33, 97 and 116. The results clearly suggest that bile acid adenylates in vivo may act as active intermediates to produce covalently bound bile acid adducts with peptides and proteins by nucleophilic displacement of the 5'-adenylic acid through the free amino groups.     

酶促合成肽的研究 II: 含羧基侧链氨基酸的肽的酶促合成

用嗜热菌酶酶促合成了一系列含门冬氨酸、谷氨酸的肽. 实验证明, 缩合反应中羧基组份中的门冬氨酸β-羧基或谷氨酸γ-羧基毋需保护. 同时也研究了邻近氨基酸残基对缩合反应的影响.     

新型光学活性α-氨基磷酸及其酯合成进展

α-氨基膦酸酯是一类很重要的天然氨基酸类似物，具有新颖的化学结构和多 种生理活性。综述了近年来光学活性α-氨基膦酸及酯的合成进展。     

氨基苯甲酸异构体在水-乙醇混合溶剂中的离解焓和熵

利用流动混合微量热法测定298.15K时单取代氨基苯甲酸邻,间,对三个异构体在水-乙醇混合溶剂中两个官能团分步离解的离解焓,结合相应的Gibbs自由能数据计算离解熵,从溶剂效应和取代基位置效应探讨其离解热力学行为。     

手性卟啉化合物聚集体与DNA 的相互作用: 电子吸收光谱 和圆二色光谱研究

使用电子吸收光和圆二色(circulardichroism,CD)光谱研究了手性氨基酸卟啉锌配合物(Thr---TPPZN)聚集体与DNA之间的相互作用,这种螺旋结构的手性卟啉聚集体能与DNA结合,L－Thr----TPPZN聚集体与DNA作用量是通过氨基酸残基与DNA的磷酸链形成氢键,结合模式为外部结合,而D----Thr--TPPZN聚集体与DNA作用除了存在以上这种氢键作用之外,卟啉单元还能部分地插入DNA中,与DNA的碱基对形成π-π堆积作用。L--Thr---TPPZN和D--Thr--TPPZn聚集体与DNA结合模式不同是由于L-------Thr----TPPZn聚集体的左手螺旋结构与DNA的右手螺旋结构不匹配,而右手螺旋结构的D--Thr-----TPPZN聚集体能嵌入同样是右手螺旋结构的DNA中。     

氨基酸在水或氯化钾水溶液中体积性质的研究

在298.15K和常压下利用密度法测定了十几种α-氨基酸在水或氯化钾水溶液中的表观摩尔体积, 由此得到了各氨基酸的偏摩尔体积和其相应的转移偏摩尔体积,以及氨基酸中侧链对其偏摩尔体积的贡献值。从理论上计算了各氨基酸在上述溶液浓度的增加而增大; 氨基酸共溶质间的相互作用以1:1形式为主; 氨基酸的水化数随盐浓度的增加而减小。对所得结果进行了定性讨论。     

The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

ABSTRACT: BACKGROUND: It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 muM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings. RESULTS: We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. CONCLUSIONS: The data outlined serves to demonstrate the "push" mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable that kinase enzymes achieve the observed 2,500-fold variation in KM through a combination of the various conserved "push" and "pull" mechanisms associated with the release of C8-H, the proton transfer cascades unique to the class of kinase in question and the resultant/concomitant creation of a pentavalent species from the gamma-phosphate group of ATP. Also demonstrated is the interplay between the role of the C8-H of ATP and the ATP concentration in the observed enzyme activity. The lability of the C8-H mediated by active site residues co-ordinated to the purine ring of ATP therefore plays a significant role in explaining the broad KM range associated with kinase steady state enzyme activities.     

ADENYLATE KINASE BOUND TO THE CHROMATOPHORE MEMBRANES OF Rhodobactor spheroides G1C

Transformation of adenylates (AMP, ADP and ATP) by washed chromatophore membranes of Rhodobactor spheroides G1C in the dark and in the light indicated the functions of ATPase (ADP + Pi in equilibrium ATP) and of an adenylate kinase (2ADP in equilibrium AMP + ATP). The activity of adenylate kinase of the chromatophores was not inhibited by AP5A, and persisted even after sonication in the presence of EDTA or CaCl2; the results suggested the presence of an adenylate kinase bound to the chromatophore membrane. In search of the enzyme, the supernatant after sonication of the chromatophores in the presence of EDTA was subjected to a molecular sieve and then to ion-exchange HPLC; a fraction with high specific adenylate kinase activity, containing a very sharp peak at 55 kDa, was isolated. Preliminary characterization indicated that it is different from the well-documented water-soluble 33 kDa adenylate kinase.     

天花粉蛋白的化学 III:溴化氰降解片段CBa的氨基酸顺序

The results from the study on the separation, purification, amino acid composition and amino acid sequence of CBa, one of the four CNBr degradation fragments of crystalline trichosanthin, are presented. Its amino acid composition is: Asp3, Thr2, Ser2, Hse1, Glu2, Gly2, Ala6, Val1, Tyr3, Phe3, Lys2, Arg1. The sequence of the CBa is Gly-Tyr-Arg-Ala-Gly-Asp-Thr-Ser- Tyr-Phe-Phe-Asn-Glu-Ala-Ser-Ala-Thr-Glu-Ala-Ala-Lys-Tyr-Val- Phe-Lys-Asp-Ala-Hso.