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.     

Role of arginine-tRNA in protein degradation by the ubiquitin pathway

Degradation of intracellular proteins through the ubiquitin and ATP-dependent proteolysis pathway involves several steps. Initially, ubiquitin is covalently linked to the proteolytic substrate in an ATP-requiring reaction. Proteins marked by ubiquitin may then be selectively lysed in a reaction that also requires ATP (for reviews see refs 1-3). A major question concerns the structural features of a protein that make it a specific substrate for ubiquitin-mediated degradation. It was shown that a free alpha-NH2 group is one important feature of the protein structure recognized by the ubiquitin ligation system, and that the half-life in vivo of a protein with an exposed amino terminus depends on its amino terminal residue. We have previously demonstrated that transfer RNA (tRNA) is essential for conjugation of ubiquitin and for the subsequent degradation of proteins with acidic amino termini (aspartate or glutamate). We now show that tRNA is required for post-translational conjugation of arginine to acidic amino termini of proteins, a modification that is essential for their degradation by the ubiquitin pathway.     

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.     

Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide

Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.     

Signal transduction in bacteria

Cells display a remarkable ability to respond to small fluctuations in their surroundings. In simple microbial systems, information from sensory receptors feeds into a circuitry of regulatory proteins that transfer high energy phosphoryl groups from histidine to aspartate side chains. This phosphotransfer network couples environmental signals to an array of response elements that control cell motility and regulate gene expression.     

苯丙氨酸分子的手性对映体转变机理及水分子（簇）的催化作用

在MP2/6 311++G(3-df,2pd-)//WB97X-D/6-311++G(-d,p-)双水平研究苯丙氨酸(Phe)分子的手性对映体转变机理, 并用分子中的原子理论(AIM)分析驻点的成键特征. 结果表明： 经过羧羟基旋转、 质子迁移、 碳 碳键旋转和氨基翻转的一系列过渡态, Phe分子在质子以氨基氮为桥梁迁移的通道a和以羰基氧与氨基氮顺次为桥梁迁移的通道b内, 实现了手性对映体转变; 当2个水分子簇作为质子迁移媒介时, 在通道b中增加了质子仅以羰基氧为桥梁迁移的反应路径； 通道a具有优势, 速控步骤的内禀能垒为25971 kJ/mol, 反应的表观能垒为27026 kJ/mol； 2个水分子簇催化使速控步骤的内禀能垒降至126.47 kJ/mol, 反应的表观能垒降至80.80 kJ/mol; 考虑零点振动能后, 质子从氨基氮向羰基氧迁移的能垒消失. 即水分子（簇）催化可使Phe分子实现手性对映体转变.     

苯丙氨酸分子的手性对映体转变机理及水分子(簇)的催化作用

在MP2/6 311++G(3-df,2pd-)//WB97X-D/6-311++G(-d,p-)双水平研究苯丙氨酸(Phe)分子的手性对映体转变机理, 并用分子中的原子理论(AIM)分析驻点的成键特征. 结果表明： 经过羧羟基旋转、 质子迁移、 碳 碳键旋转和氨基翻转的一系列过渡态, Phe分子在质子以氨基氮为桥梁迁移的通道a和以羰基氧与氨基氮顺次为桥梁迁移的通道b内, 实现了手性对映体转变; 当2个水分子簇作为质子迁移媒介时, 在通道b中增加了质子仅以羰基氧为桥梁迁移的反应路径; 通道a具有优势, 速控步骤的内禀能垒为25971 kJ/mol, 反应的表观能垒为27026 kJ/mol; 2个水分子簇催化使速控步骤的内禀能垒降至126.47 kJ/mol, 反应的表观能垒降至80.80 kJ/mol; 考虑零点振动能后, 质子从氨基氮向羰基氧迁移的能垒消失. 即水分子（簇）催化可使Phe分子实现手性对映体转变.     

Structure of human pancreatic lipase

Pancreatic lipase (triacylglycerol acyl hydrolase) fulfills a key function in dietary fat absorption by hydrolysing triglycerides into diglycerides and subsequently into monoglycerides and free fatty acids. We have determined the three-dimensional structure of the human enzyme, a single-chain glycoprotein of 449 amino acids, by X-ray crystallography and established its primary structure by sequencing complementary DNA clones. Enzymatic activity is lost after chemical modification of Ser 152 in the porcine enzyme, indicating that this residue is essential in catalysis, but other data are more consistent with a function in interfacial recognition. Our structural results are evidence that Ser 152 is the nucleophilic residue essential for catalysis. It is located in the larger N-terminal domain at the C-terminal edge of a doubly wound parallel beta-sheet and is part of an Asp-His-Ser triad, which is chemically analogous to, but structurally different from, that in the serine proteases. This putative hydrolytic site is covered by a surface loop and is therefore inaccessible to solvent. Interfacial activation, a characteristic property of lipolytic enzymes acting on water-insoluble substrates at water-lipid interfaces, probably involves a reorientation of this flap, not only in pancreatic lipases but also in the homologous hepatic and lipoprotein lipases.     

几种稳定构型精氨酸分子的手性转变反应机理及水溶剂化效应

采用密度泛函理论的B3LYP方法、微扰理论的MP2方法和自洽反应场(SCRF)理论的smd模型方法,研究了2种最稳定构型的精氨酸分子的手性转变机理及水溶剂化效应.研究发现标题反应有3条通道a、b和c.对于构型1,分别是手性碳上的质子以氨基、羰基和氨基联合以及羧基和氨基联合为桥迁移.对于构型2,分别是手性碳上的质子只以氨基为桥、羧基异构后再以氨基为桥迁移及以羧基和氨基联合作桥迁移.势能面计算表明:构型1的主反应通道都是a,决速步自由能垒分别为268.2kJ·mol~(-1),来源于质子从手性碳向氨基氮迁移的过渡态.构型2的主反应通道是b,决速步自由能垒为239.3kJ·mol~(-1),来源于质子从手性碳向氨基氮迁移的过渡态.水溶剂效应使构型2的主反应通道决速步自由能垒降到95.7kJ·mol~(-1).结果表明:随着温度的升高,构型2先手性转变;水溶剂对精氨酸的手性转变有极好的催化作用.     

Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate.     

基于氨基作为质子转移桥梁的赖氨酸分子手性转变机理

采用密度泛函理论的B3LYP方法和微扰理论的MP2方法, 考察赖氨酸分子基于氨基作为质子转移桥梁的手性转变机理以及水分子和羟基自由基对氢迁移反应的催化作用. 结果表明, 赖氨酸分子手性转变有2个通道a和b, 通道a为主反应通道, 决速步骤裸反应Gibbs自由能垒为252.6 kJ/mol, 2个水分子构成的链以及羟基自由基和水分子构成的链使通道a决速步骤的自由能垒分别降为119.5,98.5 kJ/mol. 表明水分子和羟自由基对H迁移反应有较好的催化作用, 生命体内的羟基自由基是导致左旋赖氨酸旋光异构的主要原因.     

Roles of Phe58 residue in stabilizing structure of cytochrome b5

To understand effect of (-stacking interactions between the side chain of aromatic amino acids and the porphyrin ring on structures and properties in cytochrome b5 (cyt b5), the Phe58 residue was mutated to tyrosine and tryptophan, respectively by site-directed mutagenesis. The denaturation of cyt b5 F58W and F58Y toward guanidine hydrochloride was examined by UV-visible and fluorescence spectroscopy. The kinetics of heme transfer reactions between apo-myoglobin and the mutants were studied. The results indicated that the mutation of F58 residue for Y58 or W58 reduced the interaction between of peptide and the heme group, resulting in decrease of the Tm and Cm values of the proteins, increase of the heme transfer reaction rate, and shifts of the redox potential.     

Saccharomyces cerevisiae THI4p is a suicide thiamine thiazole synthase

Thiamine pyrophosphate 1 is an essential cofactor in all living systems. Its biosynthesis involves the separate syntheses of the pyrimidine 2 and thiazole 3 precursors, which are then coupled. Two biosynthetic routes to the thiamine thiazole have been identified. In prokaryotes, five enzymes act on three substrates to produce the thiazole via a complex oxidative condensation reaction, the mechanistic details of which are now well established. In contrast, only one gene product is involved in thiazole biosynthesis in eukaryotes (THI4p in Saccharomyces cerevisiae). Here we report the preparation of fully active recombinant wild-type THI4p, the identification of an iron-dependent sulphide transfer reaction from a conserved cysteine residue of the protein to a reaction intermediate and the demonstration that THI4p is a suicide enzyme undergoing only a single turnover.     

N-二异丙氧磷酰化壳寡糖的合成与表征

通过壳寡糖和二异丙基亚磷酸酯在一种温和条件下发生Atherton-Todd反应,成功制备出了一种新型的壳寡糖含磷衍生物N-二异丙氧磷酰化壳寡糖.探讨了水与无水乙醇的体积比、壳寡糖与三乙胺的摩尔比、壳寡糖与二异丙基亚磷酸酯的摩尔比以及反应时间等因素对产品含磷量的影响.最后,对最优条件下合成的产品进行IR、1H-NMR、13C-NMR以及31P-NMR的表征,证明二异丙氧磷酰基已经成功嫁接到了壳寡糖的氨基上.这种简单而有效的方法为N-磷酰化壳聚糖衍生物的制备提供了一条新的途径.     

固—液相转移条件下前手性酮的不对称还原

在固－液相转移（ＰＴＣ）条件下,研究了前手性酮的不对称硼氢化还原,对不同的催化剂,反应溶剂、温度及反应底物对不对称诱导的影响进行了研究,结果表明固－液ＰＴＣ比液－液ＰＴＣ具有更高的光学产率。     

Visualizing molecular juggling within a B12-dependent methyltransferase complex

Derivatives of vitamin B(12) are used in methyl group transfer in biological processes as diverse as methionine synthesis in humans and CO(2) fixation in acetogenic bacteria. This seemingly straightforward reaction requires large, multimodular enzyme complexes that adopt multiple conformations to alternately activate, protect and perform catalysis on the reactive B(12) cofactor. Crystal structures determined thus far have provided structural information for only fragments of these complexes, inspiring speculation about the overall protein assembly and conformational movements inherent to activity. Here we present X-ray crystal structures of a complete 220?kDa complex that contains all enzymes responsible for B(12)-dependent methyl transfer, namely the corrinoid iron-sulphur protein and its methyltransferase from the model acetogen Moorella thermoacetica. These structures provide the first three-dimensional depiction of all protein modules required for the activation, protection and catalytic steps of B(12)-dependent methyl transfer. In addition, the structures capture B(12) at multiple locations between its 'resting' and catalytic positions, allowing visualization of the dramatic protein rearrangements that enable methyl transfer and identification of the trajectory for B(12) movement within the large enzyme scaffold. The structures are also presented alongside in crystallo spectroscopic data, which confirm enzymatic activity within crystals and demonstrate the largest known conformational movements of proteins in a crystalline state. Taken together, this work provides a model for the molecular juggling that accompanies turnover and helps explain why such an elaborate protein framework is required for such a simple, yet biologically essential reaction.     

磺胺醋酰合成技术研究

用相转移催化法合成磺胺醋酰,以TEBA(三乙基卞基氯化铵)为相转移催化剂,应用正交试验优化反应条件,考察了温度、相转移催化剂对反应的影响。收率由58.3%提高到71.67%,降低了成本,经改进纯制方法,减少了污染。     

三乙醇胺催化下的反常Reimer－Tiemann反应

本文对三乙醇胺作相转移催化剂存在下的Ｒｅｉｍｅｒ—Ｔｉｅｍａｎｎ（以下简称Ｒ—Ｔ）反应进行了研究，结果表明，三乙醇胺对此反应有明显的影响，对位和邻位产物的产率是反常的。在此催化剂存在下的反常Ｒ—Ｔ反应未见报道。     

三乙醇胺催化下的反常Reimer－Tiemann反应

本文对三乙醇胺作相转移催化剂存在下的Ｒｅｉｍｅｒ—Ｔｉｅｍａｎｎ（以下简称Ｒ—Ｔ）反应进行了研究，结果表明，三乙醇胺对此反应有明显的影响，对位和邻位产物的产率是反常的。在此催化剂存在下的反常Ｒ—Ｔ反应未见报道。