共查询到20条相似文献，搜索用时 62 毫秒
1.
Xie L Miller LM Chatterjee C Averin O Kelleher NL van der Donk WA 《Science (New York, N.Y.)》2004,303(5658):679-681
The lantibiotic lacticin 481 is synthesized on ribosomes as a prepeptide (LctA) and posttranslationally modified to its mature form. These modifications include dehydration of serines and threonines, followed by intramolecular addition of cysteines to the unsaturated amino acids, which generates cyclic thioethers. This process breaks eight chemical bonds and forms six newbonds and is catalyzed by one enzyme, LctM. We have characterized the in vitro activity of LctM, which completely processed a series of LctA mutants, displaying a permissive substrate specificity that holds promise for antibiotic engineering. 相似文献
2.
Different types of cell behavior, including growth, motility, and navigation, require actin proteins to assemble into filaments. Here, we describe a biochemical process that was able to disassemble actin filaments and limit their reassembly. Actin was a specific substrate of the multidomain oxidation-reduction enzyme, Mical, a poorly understood actin disassembly factor that directly responds to Semaphorin/Plexin extracellular repulsive cues. Actin filament subunits were directly modified by Mical on their conserved pointed-end, which is critical for filament assembly. Mical posttranslationally oxidized the methionine 44 residue within the D-loop of actin, simultaneously severing filaments and decreasing polymerization. This mechanism underlying actin cytoskeletal collapse may have broad physiological and pathological ramifications. 相似文献
3.
Redesigning trypsin: alteration of substrate specificity 总被引：40，自引：0，他引：40
C S Craik C Largman T Fletcher S Roczniak P J Barr R Fletterick W J Rutter 《Science (New York, N.Y.)》1985,228(4697):291-297
A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog. 相似文献
4.
Starai VJ Celic I Cole RN Boeke JD Escalante-Semerena JC 《Science (New York, N.Y.)》2002,298(5602):2390-2392
Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes. 相似文献
5.
L W Hardy J S Finer-Moore W R Montfort M O Jones D V Santi R M Stroud 《Science (New York, N.Y.)》1987,235(4787):448-455
The atomic structure of thymidylate synthase from Lactobacillus casei was determined at 3 angstrom resolution. The native enzyme is a dimer of identical subunits. The dimer interface is formed by an unusual association between five-stranded beta sheets present in each monomer. Comparison of known sequences with the Lactobacillus casei structure suggests that they all have a common core structure around which loops are inserted or deleted in different sequences. Residues from both subunits contribute to each active site. Two arginine side chains can contribute to binding phosphate on the substrate. The side chains of several conserved amino acids can account for other determinants of substrate binding. 相似文献
6.
Macbeth MR Schubert HL Vandemark AP Lingam AT Hill CP Bass BL 《Science (New York, N.Y.)》2005,309(5740):1534-1539
We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1. 相似文献
7.
Characterization by tandem mass spectrometry of structural modifications in proteins 总被引：10，自引：0，他引：10
Tandem mass spectrometry can be used to solve a number of protein structural problems that are not amenable to conventional methods for amino acid sequencing. Typical problems that use this approach involve characterization of peptides with blocked amino termini or peptides that have been otherwise posttranslationally processed, such as, by phosphorylation or sulfation. The structure and homogeneity of synthetic peptides can also be evaluated. Since peptides can be selectively characterized in the presence of other peptides or contaminants, the need for extensive purification is reduced or eliminated. 相似文献
8.
纤维素酶活性改良的研究进展 总被引：2，自引：1，他引：2
纤维素酶对纤维素高效水解起重要作用,但目前的纤维素酶存在活性不高、对天然木质纤维素分解能力弱等因素限制其推广和应用等问题。本文对各种改良纤维素酶活性的方法进行总结,例如利用纤维素酶和底物性质、定向诱导、直接进化、化学物理诱变育种等方法实现纤维素酶性质的改良。对每种方法的优缺点进行比较分析,其中定向诱变技术提高酶活上,获得所需的功能酶都需要耗费大量的时间和精力,而利用直接进化的方法,不需要了解蛋白质结构和酶与底物之间的关系就能够成功筛选高活性的酶,此外,根据目标酶的性质和底物的特性进行筛选也具有其独特的优势。 相似文献
9.
Enzyme action: comparison on soluble and insoluble substrate 总被引：1，自引：0，他引：1
The action of trypsin on gelatin solution is compared with its action on swollen gel microspheres in suspension. Both the solution gelatin and gel spheres, which are readily permeable to the enzyme, follow Michaelis-Menten kinetics. The apparent rate constants for dissociation of the enzyme-substrate complexes to hydrolysis products are essentially the same for both solution gelatin and spheres, an indication that gel structure in this system has a negligible influence on reaction rate once the enzyme forms a complex with the substrate. In contrast, the Michaelis constant for the gel system is greater than that for solutions below the melting point of the gel; this difference disappears as the melting point gel is approached. 相似文献
10.
复合酶降解玉米秸秆工艺条件的研究 总被引：2，自引：0，他引：2
[目的]为了寻找玉米秸秆酶解的最佳工艺条件。[方法]采用5因素3水平的正交试验,利用复合酶进行玉米秸秆的酶解,研究降解玉米秸秆的最佳工艺条件,并利用扫描电镜观察秸秆在降解过程的形态结构变化。[结果]影响该复合酶降解玉米秸秆的因素为:pH值>底物浓度>反应时间>酶用量>反应温度。该复合酶降解玉米秸秆的最佳工艺条件为:酶用量0.20%,底物浓度5.0%,反应时间3.0 h,反应温度50℃,pH值5.0。此时,降解率最高(达27.6%)。电镜观察表明:玉米秸秆经酶解作用后表面蜡质结构被降解,内部的致密结构变得松散,出现空洞。[结论]该研究为玉米秸秆的生物利用提供了参考依据。 相似文献
11.
Copper active sites play a major role in enzymatic activation of dioxygen. We trapped the copper-dioxygen complex in the enzyme peptidylglycine-alphahydroxylating monooxygenase (PHM) by freezing protein crystals that had been soaked with a slow substrate and ascorbate in the presence of oxygen. The x-ray crystal structure of this precatalytic complex, determined to 1.85-angstrom resolution, shows that oxygen binds to one of the coppers in the enzyme with an end-on geometry. Given this structure, it is likely that dioxygen is directly involved in the electron transfer and hydrogen abstraction steps of the PHM reaction. These insights may apply to other copper oxygen-activating enzymes, such as dopamine beta-monooxygenase, and to the design of biomimetic complexes. 相似文献
12.
Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue 总被引：6，自引：0，他引：6
S P White D L Scott Z Otwinowski M H Gelb P B Sigler 《Science (New York, N.Y.)》1990,250(4987):1560-1563
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. 相似文献
13.
利用酶反应热效应检测过氧化氢酶活性 总被引：2，自引：0，他引：2
为了探索酶活性测定的新方法，降低酶活性常规测试中干扰因素的影响，提高酶活性测定的准确性，利用低电势电位差计及热电偶换能器检测过氧化氢酶活性，结果表明，测定的H2O2质量分数以9．6％为好，可以用于酶活性从0．5u至3．5u酶活力的检测，当H2O2质量分数在2．4％－7．2％时，酶反应速率与底物浓度有线性关系；当底物质量分数超过9．6％时，酶反应速率显现底物抑制的动力学特征。 相似文献
14.
Interfacial catalysis: the mechanism of phospholipase A2 总被引：16，自引：0，他引：16
D L Scott S P White Z Otwinowski W Yuan M H Gelb P B Sigler 《Science (New York, N.Y.)》1990,250(4987):1541-1546
15.
Stengel KF Holdermann I Cain P Robinson C Wild K Sinning I 《Science (New York, N.Y.)》2008,321(5886):253-256
Secretory and membrane proteins carry amino-terminal signal sequences that, in cotranslational targeting, are recognized by the signal recognition particle protein SRP54 without sequence specificity. The most abundant membrane proteins on Earth are the light-harvesting chlorophyll a/b binding proteins (LHCPs). They are synthesized in the cytoplasm, imported into the chloroplast, and posttranslationally targeted to the thylakoid membrane by cpSRP, a heterodimer formed by cpSRP54 and cpSRP43. We present the 1.5 angstrom crystal structure of cpSRP43 characterized by a unique arrangement of chromodomains and ankyrin repeats. The overall shape and charge distribution of cpSRP43 resembles the SRP RNA, which is absent in chloroplasts. The complex with the internal signal sequence of LHCPs reveals that cpSRP43 specifically recognizes a DPLG peptide motif. We describe how cpSPR43 adapts the universally conserved SRP system to posttranslational targeting and insertion of the LHCP family of membrane proteins. 相似文献
16.
几丁质酶中存在结合口袋及水解活性位点，关键氨基酸与底物几丁质的结合可调控酶的水解活性。以嗜热几丁质酶Chi304为研究材料，采用同源建模分析其高级结构，发现在其底物结合口袋附近存在2个色氨酸（W140与W272），将其定点突变为丙氨酸后，采用高效液相色谱检测酶的水解产物，并进一步分析水解产物三乙酰壳三糖（DP3）与二乙酰壳二糖（DP2）的比例（DP3/DP2），用于评估突变体的效果。结果表明，突变体W140A、W272A与W140/272A水解胶体几丁质产物DP3/DP2分别较野生型提升23.3%、45.7%与80.0%。由此表明，W140与W272是影响酶与底物结合的关键氨基酸，将其突变为丙氨酸可提升Chi304的内切活力，降低外切活力。 相似文献
17.
酶法水解杂细胞最适条件及酶回收利用 总被引：1，自引：1，他引：0
研究了纤维素酶水解杂细胞的适宜条件及酶回收利用技术。在底物浓度为 3 % ,酶用量为 12 5IU/ g杂细胞(干重 ) ,酶解时间为 2 4h的最适酶解条件下 ,酶解转化率达 70 2 0 % ,还原糖浓度为 2 2 18mg/mL。用底物吸附法可以对同一批纤维素酶重复利用 3次 ,相当于每克杂细胞 (干重 )的酶用量仅为 41IU ,显著降低了纤维素酶用量 ,并有较高的酶解转化率。采用本工艺 ,1t杂细胞可得SCP饲料 2 5 0kg(干重 ) ,其粗蛋白含量为 45 5 %。 相似文献
18.
Triggering of allostery in an enzyme by a point mutation: ornithine transcarbamoylase 总被引：5，自引：0，他引：5
The origin of allostery is an unanswered question in the evolution of complex regulatory proteins. Anabolic ornithine transcarbamoylase, a trimer of identical subunits, is not an allosteric enzyme per se. However, when the active-site residue arginine-106 of the Escherichia coli enzyme is replaced with a glycine through site-directed mutagenesis, the resultant mutant enzyme manifests substrate cooperativity that is absent in the wild-type enzyme. Both homotropic and heterotropic interactions occur in the mutant enzyme. The initial velocity saturation curves of the substrates, carbamoyl phosphate and L-ornithine, conform to the Hill equation. The observed cooperativity depends on substrate but not enzyme concentration. The finding underscores the possibility that a single mutation of the enzyme in the cell could turn transcarbamoylation into a regulatory junction in the biosynthesis of L-arginine and urea. 相似文献
19.
Anatomy of a conformational change: hinged "lid" motion of the triosephosphate isomerase loop 总被引：14，自引：0，他引：14
Triosephosphate isomerase (TIM) is used as a model system for the study of how a localized conformational change in a protein structure is produced and related to enzyme reactivity. An 11-residue loop region moves more than 7 angstroms and closes over the active site when substrate binds. The loop acts like a "lid" in that it moves rigidly and is attached by two hinges to the remainder of the protein. The nature of the motion appears to be built into the loop by conserved residues; the hinge regions, in contrast, are not conserved. Results of molecular dynamics calculations confirm the structural analysis and suggest a possible ligand-induced mechanism for loop closure. 相似文献
20.
Peptidoglycan glycosyltransferases (GTs) catalyze the polymerization step of cell-wall biosynthesis, are membrane-bound, and are highly conserved across all bacteria. Long considered the "holy grail" of antibiotic research, they represent an essential and easily accessible drug target for antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus. We have determined the 2.8 angstrom structure of a bifunctional cell-wall cross-linking enzyme, including its transpeptidase and GT domains, both unliganded and complexed with the substrate analog moenomycin. The peptidoglycan GTs adopt a fold distinct from those of other GT classes. The structures give insight into critical features of the catalytic mechanism and key interactions required for enzyme inhibition. 相似文献