Chaos in an enzyme reaction

Dynamic systems are usually thought to have either monotonic or periodic behaviour. Although the possibility of other types of behaviour has been recognised for many years, the existence of non-monotonic, non-periodic behaviour in dynamic systems has been firmly established only recently. It is termed chaotic behaviour. A review on the rapidly expanding literature on chaos in discrete model systems described by difference equations has been published by May. R?ssler, on the other hand, has discussed a few published works on systems of differential equations with chaotic solutions, and he has proposed a three-component chemical model system which he argues has chaotic solutions [figure see text]. The argument is based on a theorem by Li and Yorke. Here we report the finding of chaotic behaviour as an experimental result in an enzyme system (peroxidase). Like R?ssler we base our identification of chaos on the theorem by Li and Yorke.     

Reconstruction of an enzyme by domain substitution effectively switches substrate specificity

The polar domains of the two transcarbamoylases, aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase, (OTCase) from Escherichia coli bind the common substrate carbamoyl phosphate and share extensive amino-acid sequence homology. The equatorial domains of the two enzymes differ in their substrate specificity (ATCase binds aspartate, OTCase binds ornithine) and have decreased sequence identity. While addressing the conservation of specific protein interactions during the evolution of these enzymes, we were able to switch one of their amino-acid-specific equatorial domains to produce a viable chimaeric enzyme. This was achieved by the in vitro fusion of DNA encoding the polar domain of OTCase to DNA encoding the equatorial domain of ATCase. The resulting gene fusion successfully transformed an argI-pyrB deletion strain of E. coli to pyrimidine prototrophy, giving rise to Pyr+ transformants that expressed ATCase but not OTCase activity. The formation of this active chimaeric enzyme shows that by exchanging protein domains between two functionally divergent enzymes we have achieved a switching in substrate specificity.     

Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template

Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3' end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA. We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5'-triphosphates. A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP. Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3' end of the tRNA.     

Calculation of electrostatic potentials in an enzyme active site

To be able to calculate the contributions of individual amino acids to the electrostatic field of a protein would be of considerable value in designing proteins of enhanced or altered function and stability. Recent studies on the serine protease subtilisin provide direct measurements of the electrostatic potential in the active site of the enzyme produced by two charged amino acids. We have used these results to test a recently developed method for the calculation of electrostatic interactions between two specific sites on a protein. The extent of agreement between the theoretical and experimental results suggests that the continuum solvent model used in the calculations reproduces the essential features of the interaction.     

Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon

Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical β-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical β-carbonic anhydrases and the formation of a single 15-?-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient β-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.     

Intrinsic dynamics of an enzyme underlies catalysis

A unique feature of chemical catalysis mediated by enzymes is that the catalytically reactive atoms are embedded within a folded protein. Although current understanding of enzyme function has been focused on the chemical reactions and static three-dimensional structures, the dynamic nature of proteins has been proposed to have a function in catalysis. The concept of conformational substates has been described; however, the challenge is to unravel the intimate linkage between protein flexibility and enzymatic function. Here we show that the intrinsic plasticity of the protein is a key characteristic of catalysis. The dynamics of the prolyl cis-trans isomerase cyclophilin A (CypA) in its substrate-free state and during catalysis were characterized with NMR relaxation experiments. The characteristic enzyme motions detected during catalysis are already present in the free enzyme with frequencies corresponding to the catalytic turnover rates. This correlation suggests that the protein motions necessary for catalysis are an intrinsic property of the enzyme and may even limit the overall turnover rate. Motion is localized not only to the active site but also to a wider dynamic network. Whereas coupled networks in proteins have been proposed previously, we experimentally measured the collective nature of motions with the use of mutant forms of CypA. We propose that the pre-existence of collective dynamics in enzymes before catalysis is a common feature of biocatalysts and that proteins have evolved under synergistic pressure between structure and dynamics.     

填充柱气相色谱法测定复方牙痛酊中甲醇含量

目的:建立填充柱气相色谱法测定复方牙痛酊中甲醇含量的测定方法。方法:色谱柱:PQ柱(2m×3mm;60～80m,Porakpak填料),以正丙醇为内标,进样口温度:240℃,柱温:140℃,检测器:250℃,进样量2μL。结果:甲醇在0.008～0.8μL.ml-1浓度范围内相应线性关系良好(r=0.999 6);平均回收率为93.3%,RSD=1.92%。稳定性RSD=3.45%。结论:方法操作简单,可准确用于产品的质量控制。     

超临界甲醇法制备生物柴油过程的热力学行为

超临界甲醇法制备生物柴油的过程以其显著的优点备受研究者关注,而其操作条件的改善更是研究重点．为了控制超临界甲醇法制备生物柴油过程的操作条件,需要预测超临界甲醇一油脂二元系统的临界参数．文中采用C-G基团贡献法和L—B混合规则对豆油-甲醇二元系的临界参数进行理论计算,与实验结果比较,平均误差为1．7％．此外,利用溶解度参数概念计算了不同体系条件下甲醇和油脂组分的溶解度参数,并将二者的溶解度参数差和体系的相态联系起来,从而讨论了有利于超临界酯交换反应进行的热力学条件．     

Synthesis of fast myosin induced by fast ectopic innervation of rat soleus muscle is restricted to the ectopic endplate region

Skeletal muscle fibres, long multinucleated cells, arise by fusion of mononucleated myoblasts to form a myotube that matures into the adult fibre. The two major types of mature fibre, fast and slow fibres, differ physiologically in their rate of isotonic shortening. At the molecular level these type-specific physiological properties are ascribed to different isoforms of myosin, a major protein involved in shortening. Differentiation of fast and slow fibres seems to be under the control of motoneurones, and mature fibres are innervated by only one motoneurone. When rat soleus muscle (SOL, a slow muscle) is dually innervated with a fast nerve, it acquires some properties of a fast muscle, that is, low sensitivity to caffeine and high glycogen content. We report here that in dually innervated soleus muscle the foreign fast nerve induces synthesis of fast isoforms of myosin, but only in the segment of the muscle fibre that is close to the foreign endplate. The localized influence of the nerve endplates suggest that factors controlling the phenotypic expression of the muscle fibre have a short range of activity.     

ASBR处理高浓度甲醇废水的研究

采用ASBR处理高浓度甲醇废水,考察了甲醇的厌氧抑制性,容积负荷和水力停留时间对COD去除率的影响,及不同负荷下甲醇代谢途径的变化.试验结果表明,甲醇浓度在3000～30000mg·L(-1)内对ASBR系统无厌氧抑制性;系统的最佳COD容积负荷为4.6～9.1㎏·m(-3)·d(-1).甲醇的代谢途径对COD的去除率...     

脂氧合酶--植物抗胁迫响应的关键酶

在植物抗胁迫响应中。脂氧合酶途径在抗性机制中非常重要．脂氧合酶是一种非血红素铁蛋白,在生物体内的主要作用是催化含有顺,顺-1,4-戊二烯结构的多不饱和脂肪酸加合氧分子,生成具有共轭双键的多不饱和脂肪酸的过氧化物及其一系列次生产物,如C3-,C6-,C9-和C12-醛、酮、醇、酸、酯等低分子量易挥发产物。它们和茉莉酸（茉莉酸甲酯）等构成植物抗性系统的信号分子诱导抗性蛋白合成及一系列抗胁迫反应．本文详述了脂氧合酶的结构、特性、作用机理、生理作用、活性测定方法和人工模拟。     

Improved conversion of methanol to single-cell protein by Methylophilus methylotrophus

The glutamate dehydrogenase gene of Escherichia coli has been cloned into broad host-range plasmids and can complement glutamate synthase mutants of Methylophilus methylotrophus. Assimilation of ammonia via glutamate dehydrogenase is more energy-efficient than via glutamate synthase, thus the recombinant organism converts more growth substrate, methanol, into cellular carbon.