General acid-base catalysis in the mechanism of a hepatitis delta virus ribozyme

Many protein enzymes use general acid-base catalysis as a way to increase reaction rates. The amino acid histidine is optimized for this function because it has a pK(a) (where K(a) is the acid dissociation constant) near physiological pH. The RNA enzyme (ribozyme) from hepatitis delta virus catalyzes self-cleavage of a phosphodiester bond. Reactivity-pH profiles in monovalent or divalent cations, as well as distance to the leaving-group oxygen, implicate cytosine 75 (C75) of the ribozyme as the general acid and ribozyme-bound hydrated metal hydroxide as the general base in the self-cleavage reaction. Moreover, C75 has a pK(a) perturbed to neutrality, making it "histidine-like." Anticooperative interaction is observed between protonated C75 and a metal ion, which serves to modulate the pK(a) of C75. General acid-base catalysis expands the catalytic repertoire of RNA and may provide improved rate acceleration.     

Interaction of brain synaptic vesicles induced by endogenous Ca2+ -dependent phospholipase A2

Endogenous phospholipase A2 activity of brain synaptic vesicles was Ca2+ -dependent and was increased by prostaglandin F2 alpha, calmodulin, adenosine 3', 5' -monophosphate, and adenosine triphosphate, whereas the activity was inhibited by prostaglandin E2 in the absence or presence of calmodulin. Light-scattering measurements demonstrated that stimulation of the enzyme's activity correlated with the induction of vesicle-vesicle aggregation. The effects of these compounds on endogenous synaptic vesicle phospholipase A2 activity may imply a common end point of their purported neuromodulatory actions, and indicate that synaptic vesicle phospholipase A2 may play a central role in presynaptic neurotransmission.     

A perspective on enzyme catalysis

The seminal hypotheses proposed over the years for enzymatic catalysis are scrutinized. The historical record is explored from both biochemical and theoretical perspectives. Particular attention is given to the impact of molecular motions within the protein on the enzyme's catalytic properties. A case study for the enzyme dihydrofolate reductase provides evidence for coupled networks of predominantly conserved residues that influence the protein structure and motion. Such coupled networks have important implications for the origin and evolution of enzymes, as well as for protein engineering.     

A G protein mutant that inhibits thrombin and purinergic receptor activation of phospholipase A2

The stimulation of phospholipase A2 by thrombin and type 2 (P2)-purinergic receptor agonists in Chinese hamster ovary cells is mediated by the G protein Gi. To delineate alpha chain regulatory regions responsible for control of phospholipase A2, chimeric cDNAs were constructed in which different lengths of the alpha subunit of Gs (alpha s) were replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). When a carboxyl-terminal chimera alpha s-i(38), which has the last 38 amino acids of alpha s substituted with the last 36 residues of alpha i2, was expressed in Chinese hamster ovary cells, the receptor-stimulated phospholipase A2 activity was inhibited, although the chimera could still activate adenylyl cyclase. Thus, alpha s-i(38) is an active alpha s, but also a dominant negative alpha i molecule, indicating that the last 36 amino acids of alpha i2 are a critical domain for G protein regulation of phospholipase A2 activity.     

Structures of free and inhibited human secretory phospholipase A2 from inflammatory exudate

Phospholipase A2 (PLA2) participates in a wide range of cellular processes including inflammation and transmembrane signaling. A human nonpancreatic secretory PLA2 (hnps-PLA2) has been identified that is found in high concentrations in the synovial fluid of patients with rheumatoid arthritis and in the plasma of patients with septic shock. This enzyme is secreted from certain cell types in response to the proinflammatory cytokines, tumor necrosis factor or interleukin-1. The crystal structures of the calcium-bound form of this enzyme have been determined at physiological pH both in the presence [2.1 angstrom (A) resolution] and absence (2.2 A resolution) of a transition-state analogue. Although the critical features that suggest the chemistry of catalysis are identical to those inferred from the crystal structures of other extracellular PLA2s, the shape of the hydrophobic channel of hnps-PLA2 is uniquely modulated by substrate binding.     

苏木水提物抗蛇毒磷脂酶A2活性研究

中药苏木(Sappan Lignum)来源于植物苏木(Caesalpinia sappan L.)的干燥心材,具有止血 抗炎等功效。采用琼脂糖平板法研究不同浓度的苏木水提物（0.03g/ml,0.06g/ml,0.09g/ml, 0.12g/ml,0.15g/ml）对蛇毒sPLA2水解活性的抑制作用,测量透明圈和孔的直径,根据酶活标准曲 线计算其抑制率。结果表明0.15g/ml 苏木水提物对五步蛇毒sPLA2水解活性有抑制作用。     

意大利蜜蜂蜂毒磷脂酶A2基因在大肠杆菌中的表达

将意大利蜜蜂蜂毒磷脂酶A2(AmPLA2)成熟肽编码区基因(405 bp) 插入表达载体pETBlue-1,在大肠杆菌Tuner(DE3)placⅠ中诱导表达,经SDS-PAGE电泳检测,表达产物分子量为15 kD,约占细菌总蛋白的4.3%;用兔抗AmPLA2多克隆血清为第一抗体作Western blot检测,表达产物显示与天然AmPLA2同样的特异性印迹,证实AmPLA2 基因已在大肠杆菌中得到表达.     

Crystal structure of bee-venom phospholipase A2 in a complex with a transition-state analogue

The 2.0 angstroms crystal structure of a complex containing bee-venom phospholipase A2 (PLA2) and a phosphonate transition-state analogue was solved by multiple isomorphous replacement. The electron-density map is sufficiently detailed to visualize the proximal sugars of the enzyme's N-linked carbohydrate and a single molecule of the transition-state analogue bound ot its active center. Although bee-venom PLA2 does not belong to the large homologous Class I/II family that encompasses most other well-studied PLA2s, there is segmental sequence similarity and conservation of many functional substructures. Comparison of the bee-venom enzyme with other phospholipase structures provides compelling evidence for a common catalytic mechanism.     

Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue

The crystal structure of a complex between a phosphonate transition-state analogue and the phospholipase A2 (PLA2) from Naja naja atra venom has been solved and refined to a resolution of 2.0 angstroms. The identical stereochemistry of the two complexes that comprise the crystal's asymmetric unit indicates both the manner in which the transition state is stabilized and how the hydrophobic fatty acyl chains of the substrate are accommodated by the enzyme during interfacial catalysis. The critical features that suggest the chemistry of binding and catalysis are the same as those seen in the crystal structure of a similar complex formed with the evolutionarily distant bee-venom PLA2.     

Enhanced activity and altered specificity of phospholipase A2 by deletion of a surface loop

Protein engineering and x-ray crystallography have been used to study the role of a surface loop that is present in pancreatic phospholipases but is absent in snake venom phospholipases. Removal of residues 62 to 66 from porcine pancreatic phospholipase A2 does not change the binding constant for micelles significantly, but it improves catalytic activity up to 16 times on micellar (zwitterionic) lecithin substrates. In contrast, the decrease in activity on negatively charged substrates is greater than fourfold. A crystallographic study of the mutant enzyme shows that the region of the deletion has a well-defined structure that differs from the structure of the wild-type enzyme. No structural changes in the active site of the enzyme were detected.     

A single-molecule study of RNA catalysis and folding

Using fluorescence microscopy, we studied the catalysis by and folding of individual Tetrahymena thermophila ribozyme molecules. The dye-labeled and surface-immobilized ribozymes used were shown to be functionally indistinguishable from the unmodified free ribozyme in solution. A reversible local folding step in which a duplex docks and undocks from the ribozyme core was observed directly in single-molecule time trajectories, allowing the determination of the rate constants and characterization of the transition state. A rarely populated docked state, not measurable by ensemble methods, was observed. In the overall folding process, intermediate folding states and multiple folding pathways were observed. In addition to observing previously established folding pathways, a pathway with an observed folding rate constant of 1 per second was discovered. These results establish single-molecule fluorescence as a powerful tool for examining RNA folding.     

Heterogeneous catalysis: some recent developments

In recent years major progress has been made in the area of heterogeneous catalysis by metals. Much has been learned about the nature of metal catalysts and of catalytic phenomena on metals. Characteristic patterns of catalytic behavior among the metallic elements have been established for certain classes of reactions, and these patterns provide a first step toward a more comprehensive understanding of catalytic specificity. Studies on metal alloys and related bimetallic catalysts have revived interest in a geometric factor in surface catalysis to complement the traditional electronic factor. Closely related to this geometric factor is the discovery that selectivity, rather than activity alone, is a major factor in reactions on bimetallic catalysts. Concurrent with progress in understanding how catalysts work, advances are also being made in the development of new catalyst systems, examples of which are the bimetallic (or polymetallic) cluster catalysts. Research in this area provides an example of how advances in catalyst technology can be realized within a framework of fundamental research on catalytic phenomena (38).     

A glycan-phosphatidylinositol-specific phospholipase D in human serum

A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.     

步长脑心通胶囊对急性脑梗死患者血浆脂蛋白磷酯酶A2水平的影响

目的 观察步长脑心通胶囊对急性脑梗死患者不同时期血浆脂蛋白磷脂酶A2(Lp-PLA2)水平的影响.方法116例急性脑梗死患者随机分为对照组(56例)和观察组(60例),对照组采用常规疗法,观察组加用步长脑心通胶囊.监测两组患者治疗后1、2、4、8、12周的血浆Lp-PLA2水平.结果 治疗后两组上述观察时期血浆Lp-PLA2水平均低于治疗前,且观察组显著低于对照组,差异均有统计学意义(P＜0.01).结论 步长脑心通胶囊治疗急性脑梗死可降低血浆Lp-PLA2水平.     

Perovskite oxides: materials science in catalysis

In a time of growing need for catalysts, perovskites have been rediscovered as a family of catalysts of such great diversity that a broad spectrum of scientific disciplines have been brought to bear in their study and application. Because of the wide range of ions and valences which this simple structure can accommodate, the perovskites lend themselves to chemical tailoring. It is relatively simple to synthesize perovskites because of the flexibility of the structure to diverse chemistry. Many of the techniques of ceramic powder preparation are applicable to perovskite catalysts. In their own right, they are therefore of interest as a model system for the correlation of solid-state parameters and catalytic mechanisms. Such correlations [See figure in the PDF file] have recently been found between the rate and selectivity of oxidation-reduction reactions and the thermodynamic and electronic parameters of the solid. For commercial processes such as those mentioned in the introduction, perovskite catalysts have not yet proven to be practical. Much of the initial interest in these catalysts related to their use in automobile exhaust control. Current interest in this field centers on noble metalsubstituted perovskites resistant to S poisoning for single-bed, dual-bed, and three-way catalyst configurations. The formulations commercially tested to date have shown considerable promise, but long-term stability has not yet been achieved. A very large fraction of the elements that make up presently used commercial catalysts can be incorporated in the structure of perovskite oxides. Conversely, it is anticipated that perovskite oxides, appropriately formulated, will show catalytic activity for a large variety of chemical conversions. Even though this expectation is by no means a prediction of commercial success in the face of competition by existing catalyst systems, it makes these oxides attractive models in the study of catalytic chemical conversion. By appropriate formulation many desirable properties can be tailored, including the valence state of transition metal ions, the binding energy and diffusion of O in the lattice, the distance between active sites, and the magnetic and conductive properties of the solid. Only a very small fraction of possible perovskite formulations have been explored as catalysts. It is expected that further investigation will greatly expand the scope of perovskite catalysis, extend the understanding of solid-state parameters in catalysis, and contribute to the development of practical catalytic processes.     

Interfacial Organisms: Passive Ventilation in the Velocity Gradients near Surfaces

A variety of animals, including certain sponges, tube-dwellinlg worms, tropical termites, and prairie dogs, either are themselves arranged or construct domiciles arranged to permit flow of fluid inside the system driven by a velocity gradient in an external stream of fluid.     

Antibody catalysis of the oxidation of water

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.     

Merging photoredox catalysis with organocatalysis: the direct asymmetric alkylation of aldehydes

Photoredox catalysis and organocatalysis represent two powerful fields of molecule activation that have found widespread application in the areas of inorganic and organic chemistry, respectively. We merged these two catalysis fields to solve problems in asymmetric chemical synthesis. Specifically, the enantioselective intermolecular alpha-alkylation of aldehydes has been accomplished using an interwoven activation pathway that combines both the photoredox catalyst Ru(bpy)3Cl2 (where bpy is 2,2'-bipyridine) and an imidazolidinone organocatalyst. This broadly applicable, yet previously elusive, alkylation reaction is now highly enantioselective and operationally trivial.     

Asymmetric catalysis of the transannular Diels-Alder reaction

Transannular chemical reactions are unparalleled in their ability to generate high degrees of stereochemical and architectural complexity in a single transformation. However, the successful application of this approach in synthesis depends on the ability to predict and control the outcome of the transannular reaction. Use of a chiral catalyst in this context represents an attractive, yet unused, strategy. This report describes a catalytic, asymmetric transannnular Diels-Alder (TADA) reaction that affords polycyclic products in high enantiomeric excess. This catalyst system can also alter the inherent diastereoselectivity of cyclizations with substrates containing chiral centers. Additionally, the catalytic enantioselective TADA has been used as the key step in a total synthesis of the sesquiterpene 11,12-diacetoxydrimane; this route may provide a general approach to the polycyclic carbon framework shared by many terpene natural products.