油菜籽焦磷酸:果糖-6-磷酸1-磷酸转移酶的动力学研究

采用DEAE-Sephadex A-50及磷酸纤维素柱层析,用底物亲和洗脱法从萌发油菜(Brassica napus)种子中分离纯化了焦磷酸:果糖-6-磷酸1-磷酸转移酶(PFP).纯化倍数679.7倍,比活力为21.75 单位/毫克.蛋白,活力回收率22.9%.酶的最适pH值为7.5,Mg2+和M2+n对酶有激活作用.进行酶的初级动力学及稳态动力学研究,对果糖-6-磷酸(F6P)表现为典型的米氏规律,Km值为3.33 mmol/L;对焦磷酸(PPi)的活力变化,在PPi浓度小于1.0 mmol/L时具有部分米氏酶特点(Km＝1.0 mmol/L),大于1.0 mmol/L时,PPi对酶有抑制作用.从两底物F6P和PPi的相互作用以及产物磷酸(Pi)与底物(F6P和PPi)的相互关系分析,初步推断油菜籽PFP的催化反应为双底物双产物的有序机制.     

甘蔗果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶基因的cDNA克隆及序列分析

以高等植物玉米、水稻的果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶(F2KP)的保守区域设计引物,并采用逆转录--聚合酶链式反应(RT-PCR)方法,从甘蔗叶片中扩增F2KP的cDNA片段,命名为SoF2KP-L.该cDNA片段长2 178 bp,含2 085 bp的完整开放阅读框,编码 694个氨基酸.序列分析表明,甘蔗F2KP与其它已知的高等植物F2KP具有很高的同源性.     

嗜酸氧化亚铁硫杆菌的核糖-5-磷酸异构酶的同源建模研究

采用同源建模技术和分子动力学模拟方法,构建了嗜酸氧化亚铁硫杆菌Acidithiobacillus ferrooxidans(A.f)的核糖-5-磷酸异构酶(rpiA)基因编码的蛋白质三维分子结构模型。将结构模型进行绑定位点搜索并与底物核糖-5-磷酸(R5P)进行柔性分子对接,结果显示,R5P被招募到A.ferrooxidans的RpiA的活性位点并随后被激活;残基Asp81,Thr31,Lys121,Ser30,Glu103,Asp84,Lys94,Asp118,Lys7,Gly97,Gly29,Gly95,Thr28和H2O对底物绑定或催化起重要作用,其中,Gly97,Gly29,Gly95和Thr28是新识别的残基,它们在其他生物体的RpiA中相当保守但未被发现。     

果糖-1,6-二磷酸醛缩酶的研究进展

果糖-1,6-二磷酸醛缩酶(FBA)是糖酵解途径中一种重要的生物酶,可催化底物果糖-1,6-二磷酸裂解为3-磷酸甘油醛和磷酸二羟丙酮或反向的醛醇缩合反应.主要介绍了FBA的分类及其功能,如参与生物体内代谢、充当纤溶酶原结合蛋白、作为宿主细胞粘附结合剂、转录调节因子等,并总结了FBA在抗真菌药物靶点、抗病原体疫苗方面的应用,旨在为后续FBA的深入研究提供基础资料.     

高渗物质对α-磷酸甘油酯酶活性的影响

α-磷酸甘油酯酶是催化甘油生成的最后一步反应的酶,该酶具有高度的底物专一性.本文研究了高渗物质NaCl、KCl和甘露醇在不同浓度下对该酶活性的影响.结果表明,同一高渗介质的不同浓度对α-磷酸甘油酯酶的活性有不同程度的影响.其中氯化钠的影响程度最大,氯化钾的影响程度次之,甘露醇的影响程度最小.相同浓度的同一高渗介质,在不同的时间点上,该酶的活性大小也不相同,比酶活的峰值所对应的高渗介质的浓度随时间逐渐减小.     

菠萝焦磷酸:果糖-6-磷酸1-磷酸转移酶的分离纯化及性质研究

应用硫酸铵分级分离、DEAE-纤维素、Sephadex G-200、磷酸纤维素柱层析分离纯化了菠萝叶片焦磷酸:果糖-6-磷酸1-磷酸转移酶(PFP).凝胶过滤和非变性聚丙烯酰胺梯度电泳测定酶的分子量为132kD和140kD,SDS-聚丙烯酰胺电泳分析得到一条分子量为66kD的蛋白主带,表明该酶可能是由同种亚基组成的二聚体.此外,还对该酶的部分酶学性质进行了初步研究.     

枯草杆菌6-磷酸葡萄糖酸脱氢酶的分离纯化及部分性质研究

采用DEAE—Sephaurose离子交换层析，Blue-Seplaarose CL-6B特异结合层析和Sephadex G-200凝胶过滤技术，对枯草芽孢杆菌中的6-磷酸葡萄糖酸脱氢酶进行了分离纯化，纯化倍数为112．3倍，比活为1．46U／mg，回收率为8．2％，纯化酶液经聚丙烯酰胺凝胶电泳检验为单一带，测得全酶相对分子质量为107kD，具有两个分子量相同的亚基，亚基相对分子质量为51kD，最适pH值为8．0，最适温度为30℃，对热不稳定，以6-磷酸葡萄糖酸和NADP^ 为底物，其Km值分别为Km1(NADP^ )=19．8μmol／L和Km2(6-GPA)=22．6μmol／L，在5mmol／L～50mmol／L浓度范围内，Mg^ ，Ca^2 和Mn^2 对酶有激活作用，Fe^3 和Cu^2 对酶有抑制作用，K^ ，Na^ ，Cl^-，NO^-3，SO^-4对酶几乎没有影响，用PMSF，TNBS，NBS，DTT和BrAc对酶进行修饰，实验结果表明，丝氨酸、苏氨酸、赖氨酸和组氨酸残基可能是该酶活性中心的功能基团。     

文昌鱼酸性磷酸酯酶的化学修饰

用DEAE-纤维素(DE-22)并改进酶的提纯方法,聚丙烯酰胺凝胶电泳为单一蛋白区带的酶制剂比活力为359μM/mg(E)/min(Npp为底物)·用凝胶过滤法测得该酶分子量为58600,氨基酸组成有20种,共467个氨基酸残基。10~(-4)M pCMB使酶活力丧失98％;1×10~(-3)M PMSP该酶活力丧失96％,2×10~(-4)M NBS以及1×10~(-2)M BrAc酶活力均丧失90％·酶修饰失活呈一级反应,修饰过的酶紫外280nm吸收峰消失·初步判断文昌鱼AcPase分子上的Cys,Trp,His,Ser等氨基酸残基可能是酶的活力必需基团。     

丙酮对锯缘青蟹N-乙酰-β-D-氨基葡萄糖苷酶活力与构象的影响

以丙酮为效应物,研究其对锯缘青蟹(Scylla serrata)N-乙酰-β-D氨基葡萄糖苷酶(NAGase)活力的影响,结果表明:该酶的剩余活力随着丙酮浓度增大而呈指数下降,当丙酮浓度达25%,酶的剩余活力仅为20%,说明丙酮对青蟹NA-Gase有明显的失活作用.导致酶活力丧失50%的丙酮浓度为7.5%.在较低丙酮浓度(＜10%)的失活是可逆的反应过程.动力学研究表明,该酶的失活过程属于混合型,并进一步测定游离酶(E)和酶底物络合物(ES)与丙酮的结合常数(K1和K1s),分别为4.06%和10.49%,K1＜K1s,说明底物存在对酶被丙酮的失活作用有一定的保护作用.应用荧光发射光谱研究青蟹NAGase经丙酮微扰后的分子构象变化情况,结果表明:丙酮对酶分子构象有显著的影响,酶的内源荧光强度随丙酮浓度增大而降低,说明酶分子中的生色基团Trp和Tyr残基的微环境发生了变化.     

橡胶树糖代谢调控基因HbF2KP的克隆及表达

根据拟南芥、蓖麻及杨树果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶基因(F2KP)的核苷酸序列,使用橡胶树胶乳转录组数据库拼接到1条1 228 bp的核苷酸序列,通过两次RACE-PCR获得了橡胶树长2 629 bp的 F2KP基因cDNA序列,命名为HbF2KP。ORF Finder软件分析结果显示,该序列含有211 bp 的5’端非编码区,173 bp的3’端非编码区,以及长度为2 244 bp的开放阅读框(ORF)。氨基酸同源性分析发现,该蛋白含有保守的激酶和酯酶结构域,属于具有双功能的果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶。生物信息学分析显示,HbF2KP蛋白无导肽酶切位点,不具有跨膜结构域且属于亲水蛋白,推测该蛋白可能在细胞质中发挥作用。实时荧光定量PCR分析表明,HbF2KP基因在光合组织及非光合组织中均有表达。对非光合库组织胶乳(产胶细胞的细胞质)中的表达模式进行分析,结果显示该基因表达受割胶、伤害处理及死皮胁迫调控,推测其在橡胶树胶乳糖代谢中发挥重要的调控作用,参与橡胶烃的生物合成调控及死皮胁迫应答。此外,HbF2KP表达受部分植物激素或激素类似物如乙烯利(ET)、植物生长素(2,4-D)、脱落酸(ABA)、水杨酸(SA)及赤霉素(GA)的调控,但调控作用不明显。     

Conversion of allosteric inhibition to activation in phosphofructokinase by protein engineering

Many enzymes are subject to allosteric control, often with inhibitors and activators binding to the same effector site. Phosphofructokinase in Escherichia coli is such an enzyme, being inhibited by phosphoenolpyruvate (PEP) and activated by ADP and GDP. How do individual interactions with effectors affect the balance between activation and inhibition, especially when both ligands share aspects of the same binding site? We find that mutation of a single residue in the effector site, Glu----Ala 187, leads to PEP being an activator rather than an inhibitor. With low concentrations of the substrate fructose-6-phosphate, the mutant enzyme is more than one hundred times more active than wild-type enzyme at millimolar concentrations of PEP. The classical Monod-Wyman-Changeux two-state model is too simple to account for the properties of the mutant enzyme.     

Inhibition of Alkaline Phosphatase from Pearl Oyster Pinctada fucata by o-Phthalaldehyde： Involvement of Lysine and Histidine Residues at the Active Site

Alkaline phosphatase from Pinctada fucata was inactivated by o-phthalaldehyde （OPA）. The inactivation followed pseudo first-order kinetics with a second rate constant of 0.167 （mmol/L）^-1·min^-1 at pH 7.5 and 25℃. A Tsou＇s plot analysis showed that inactivation occurred upon formation of one isoindole group. The OPA-modified enzyme lost the ability to bind with the specific affinity column and the presence of substrates or competitive inhibitors protected the enzyme from inactivation. The results revealed that the OPA-reaction site was at the enzyme substrate binding site. Prior modification of the enzyme by lysine or histidine specific reagent abolished formation of the isoindole derivatives, suggesting that lysine and histidine residues were involved in the OPA-induced inactivation. Taken together, OPA inactivated the alkaline phosphatase from Pinctada fucata by cross-linking lysine and histidine residues at the active site and formed an isoindole group at the substrate binding site of the enzyme.     

Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase

Enzymes catalyse specific reactions and are essential for maintaining life. Although some are referred to as being bifunctional, they consist of either two distinct catalytic domains or a single domain that displays promiscuous substrate specificity. Thus, one enzyme active site is generally responsible for one biochemical reaction. In contrast to this conventional concept, archaeal fructose-1,6-bisphosphate (FBP) aldolase/phosphatase (FBPA/P) consists of a single catalytic domain, but catalyses two chemically distinct reactions of gluconeogenesis: (1) the reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) to FBP; (2) the dephosphorylation of FBP to fructose-6-phosphate (F6P). Thus, FBPA/P is fundamentally different from ordinary enzymes whose active sites are responsible for a specific reaction. However, the molecular mechanism by which FBPA/P achieves its unusual bifunctionality remains unknown. Here we report the crystal structure of FBPA/P at 1.5-? resolution in the aldolase form, where a critical lysine residue forms a Schiff base with DHAP. A structural comparison of the aldolase form with a previously determined phosphatase form revealed a dramatic conformational change in the active site, demonstrating that FBPA/P metamorphoses its active-site architecture to exhibit dual activities. Thus, our findings expand the conventional concept that one enzyme catalyses one biochemical reaction.     

Active-site mutants altering the cooperativity of E. coli phosphofructokinase

Crystal structures of the high- and low-activity states of the allosteric enzyme phosphofructokinase implicate three arginines in substrate binding, catalysis and cooperativity. Arginines 162 and 243 reach into the active site from an adjacent subunit and interact with the cooperative substrate fructose 6-phosphate. Mutation of these arginines to serine results in mutant enzymes with reduced substrate binding and lowered cooperativity, but with little change in their catalytic ability (kcat). Arg 72 bridges the two substrates fructose 6-phosphate and ATP, and interacts with the 1-phosphate of the product fructose 1,6-biphosphate. Mutation of this residue to serine reduces the catalytic activity, cooperativity and binding of fructose 6-phosphate and fructose 1,6-bisphosphate. In the reverse reaction, the kinetics of wild-type and the Ser 72 mutant with respect to fructose 1,6-bisphosphate are hyperbolic, whereas those of the Ser 162 and Ser 243 mutants are sigmoidal. These results show that each of the three arginines contributes to cooperativity and to the transmission of allosteric signals between the four subunit of the enzyme.     

Structure and control of phosphofructokinase from Bacillus stearothermophilus.

The allosteric enzyme phosphofructokinase binds its substrate fructose-6-phosphate between two subunits of the tetramer, and allosteric effectors between another pair of subunits. The effector binding site accommodates both the activator and the inhibitor. The substrate cooperativity and allosteric control are mediated by these ligand bridges between subunits.     

Site-directed mutagenesis reveals role of mobile arginine residue in lactate dehydrogenase catalysis

The binding of substrates to lactate dehydrogenases induces a marked rearrangement of the protein structure in which a 'loop' of polypeptide (residues 98-110) closes over the active site of the enzyme. In this rearrangement, arginine 109 (a basic residue conserved in all known lactate dehydrogenase sequences and in the homologous malate dehydrogenases) moves 0.8 nm from a position in the solvent to one in the active site where its guanidinium group resides within hydrogen bonding distance of both the reactive carbonyl of pyruvate and imidazole ring of the catalytic histidine 195 (see Fig. 1). Whilst this feature of the enzyme has been commented upon previously, the function of this mobile arginine residue during catalysis has not been tested experimentally. The advent of protein engineering has now enabled us to define the role of this basic residue by substituting it with the neutral glutamine. Transient kinetic and equilibrium studies of the mutant enzyme indicate that arginine 109 enhances the polarization of the pyruvate carbonyl group in the ground state and stabilizes the transition state. The gross active-site structure of the enzyme is not altered by the mutation since an alternative catalytic function of the enzyme (rate of addition of sulphite to NAD+), which does not require hydride transfer, is insensitive to the arginine----glutamine substitution.     

Active-site remodelling in the bifunctional fructose-1,6-bisphosphate aldolase/phosphatase

Fructose-1,6-bisphosphate (FBP) aldolase/phosphatase is a bifunctional, thermostable enzyme that catalyses two subsequent steps in gluconeogenesis in most archaea and in deeply branching bacterial lineages. It mediates the aldol condensation of heat-labile dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) to FBP, as well as the subsequent, irreversible hydrolysis of the product to yield the stable fructose-6-phosphate (F6P) and inorganic phosphate; no reaction intermediates are released. Here we present a series of structural snapshots of the reaction that reveal a substantial remodelling of the active site through the movement of loop regions that create different catalytic functionalities at the same location. We have solved the three-dimensional structures of FBP aldolase/phosphatase from thermophilic Thermoproteus neutrophilus in a ligand-free state as well as in complex with the substrates DHAP and FBP and the product F6P to resolutions up to 1.3??. In conjunction with mutagenesis data, this pinpoints the residues required for the two reaction steps and shows that the sequential binding of additional Mg(2+) cations reversibly facilitates the reaction. FBP aldolase/phosphatase is an ancestral gluconeogenic enzyme optimized for high ambient temperatures, and our work resolves how consecutive structural rearrangements reorganize the catalytic centre of the protein to carry out two canonical reactions in a very non-canonical type of bifunctionality.     

Inactivation of Lactate Dehydrogenase from Pig Heart by o—Phthalaldehyde

IntroductionLactate dehydrogenase( EC1 .1 .1 .2 7,LDH) playsa vital role in the energy flow of higher organisms.It is a tetrameric enzyme that catalyzes thereversible dehydrogenation of lactate,converting itto pyruvate.The enzyme exists in five isozymicforms which are not usually found all together inone tissue or organ.At least three different typesof LDH subunits( M,H and X) are known invertebrates,butthe X subunits are usually presentin only one or at mosta few tissues[1,2 ] .　Variou…     

Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosome mimicry

The bacteria causing diphtheria, whooping cough, cholera and other diseases secrete mono-ADP-ribosylating toxins that modify intracellular proteins. Here, we describe four structures of a catalytically active complex between a fragment of Pseudomonas aeruginosa exotoxin A (ETA) and its protein substrate, translation elongation factor 2 (eEF2). The target residue in eEF2, diphthamide (a modified histidine), spans across a cleft and faces the two phosphates and a ribose of the non-hydrolysable NAD+ analogue, betaTAD. This suggests that the diphthamide is involved in triggering NAD+ cleavage and interacting with the proposed oxacarbenium intermediate during the nucleophilic substitution reaction, explaining the requirement of diphthamide for ADP ribosylation. Diphtheria toxin may recognize eEF2 in a manner similar to ETA. Notably, the toxin-bound betaTAD phosphates mimic the phosphate backbone of two nucleotides in a conformational switch of 18S rRNA, thereby achieving universal recognition of eEF2 by ETA.