Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations

The crystal structure of a murine adenosine deaminase complexed with 6-hydroxyl-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, has been determined and refined at 2.4 angstrom resolution. The structure is folded as an eight-stranded parallel alpha/beta barrel with a deep pocket at the beta-barrel COOH-terminal end wherein the inhibitor and a zinc are bound and completely sequestered. The presence of the zinc cofactor and the precise structure of the bound analog were not previously known. The 6R isomer of the analog is very tightly held in place by the coordination of the 6-hydroxyl to the zinc and the formation of nine hydrogen bonds. On the basis of the structure of the complex a stereoselective addition-elimination or SN2 mechanism of the enzyme is proposed with the zinc atom and the Glu and Asp residues playing key roles. A molecular explanation of a hereditary disease caused by several point mutations of an enzyme is also presented.     

Determining transition-state geometries in liquids using 2D-IR

Many properties of chemical reactions are determined by the transition state connecting reactant and product, yet it is difficult to directly obtain any information about these short-lived structures in liquids. We show that two-dimensional infrared (2D-IR) spectroscopy can provide direct information about transition states by tracking the transformation of vibrational modes as a molecule crossed a transition state. We successfully monitored a simple chemical reaction, the fluxional rearrangement of Fe(CO)5, in which the exchange of axial and equatorial CO ligands causes an exchange of vibrational energy between the normal modes of the molecule. This energy transfer provides direct evidence regarding the time scale, transition state, and mechanism of the reaction.     

Surface science and catalysis

During the last 20 years, surface scientists have developed a variety of techniques that make it possible to study, on the molecular level, the structure, composition, and chemical bonding in the surface monolayer. Both single-crystal model catalysts and real catalyst systems that are active in important chemical processes have been investigated by a combination of ultrahigh-vacuum surface science techniques and high-pressure kinetic techniques in an effort to determine the relation between the structure and composition of the surface and the rates of reaction and selectivities. The structure of the atomic surface, a strongly adsorbed overlayer, and the oxidation states of surface atoms are the important molecular features of an active catalyst. As a consequence of modern surface science, the design and preparation of catalysts has developed from an art into a high technology.     

Observation of transition-state vibrational thresholds in the rate of dissociation of ketene

Rate constants for the dissociation of highly vibrationally excited ketene (CH(2)CO) have been measured at the threshold for the production of CH(2)((3)B(1)) and CO((1)Sigma(+)). The rate constant increases in a stepwise manner with increasing energy, consistent with the long-standing premise that the rate of a unimolecular reaction is controlled by flux through quantized transition-state thresholds. The data give the energies of the torsional and C-C-O bending vibrations of the transition state.     

Enzyme dynamics during catalysis

Internal protein dynamics are intimately connected to enzymatic catalysis. However, enzyme motions linked to substrate turnover remain largely unknown. We have studied dynamics of an enzyme during catalysis at atomic resolution using nuclear magnetic resonance relaxation methods. During catalytic action of the enzyme cyclophilin A, we detect conformational fluctuations of the active site that occur on a time scale of hundreds of microseconds. The rates of conformational dynamics of the enzyme strongly correlate with the microscopic rates of substrate turnover. The present results, together with available structural data, allow a prediction of the reaction trajectory.     

花生蛋白酶解条件及酶解产物功能特性的研究

[目的]研究花生蛋白酶解条件、酶解产物的功能特性。[方法]采用酶解法,从花生饼粕粉中提取花生蛋白,通过单因素试验,考察了不同条件下花生饼粕粉中蛋白质的提取情况,并对其功能特性进行了研究。[结果]最佳的蛋白质提取率的条件为花生饼粕粉在55℃水热处理6 h,固液比为1∶20,溶液pH为12,加酶量为0.5%,在此条件下花生蛋白的提取率为74.62%。[结论]酶解花生蛋白具有较高的提取率。酶解产物具有较好的乳化性、起泡性及保水性。     

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.     

Organometallic chemistry in homogeneous catalysis

Many reactions catalyzed by soluble transition metal compounds proceed by way of organometallic intermediates, even though the original catalyst may be a simple salt. This generality is illustrated for three industrial syntheses of acetic acid that use homogeneous catalysts. Some developments in organometallic chemistry that may extend the utility of homogeneous catalysis are photoactivation of catalysts and the recognition of the importance of metallacyclic intermediates.     

不同品种梨汁酶促褐变因子及相关性

[目的]明确不同品种梨汁酶促褐变的理化因子(相关酶活性、底物组成和含量、pH),并由相关性分析探讨梨汁酶促褐变主要原因,为品种选择和褐变控制提供理论依据和技术支持.[方法]以鸭梨、长把梨等7种梨为原料,测定梨果实中多酚氧化酶(PPO)、过氧化物酶(POD)、苯丙氨酸转氨酶(PAl.)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)等5种褐变相关酶的活性,总酚含量,梨果实的pH以及鲜榨梨汁的色值(T<,440>);用HPLC法鉴定梨果实中酚类物质的种类.[结果]供试7个品种梨汁的酶促褐变程度存在很大差别,黄冠梨和黄金梨汁褐变较轻,雪梨和水晶梨汁褐变较重.不同品种梨汁酶促褐变的主要相关酶不同,并且不同品种梨果实中的酶促褐变相关酶活性存在较大区别.梨果实的总酚含量以及与梨汁褐变的相关度因品种而异;供试的梨果实中的酚类物质主要是(+)-儿茶素、绿原酸和香豆素,且不同品种的酚类组成不同.不同品种梨果实的pH存在差异,梨汁的色值与果实的pH均呈显著相关.[结论]不同品种梨汁的酶促褐变程度有很大差别.梨果实中褐变相关酶类的活性、酚类物质含量和组成以及对梨汁酶促褐变的影响程度因品种而异,梨汁的酶促褐变与酶类的相关度高于与总酚含量的相关度.梨汁酶促褐变与梨果实的pH呈显著相关.     

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.     

Homogeneous catalysis in supercritical fluids

Supercritical fluids (SCFs), compounds heated and pressurized beyond the critical point, have many unusual properties. Homogeneous molecular catalysts, which have far greater control over selectivity than heterogeneous solid catalysts, are now being tested in SCFs, and early results show that high rates, improved selectivity, and elimination of masstransfer problems can be achieved. As industry moves away from toxic or environmentally damaging solvents, supercritical carbon dioxide may be an ideal replacement medium for nonpolar or weakly polar chemical processes. More than simply substitutes for nonpolar solvents, SCFs can radically change the observed chemistry. Supercritical carbon dioxide is also an excellent medium for its own fixation, as demonstrated by studies of its hydrogenation.     

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.