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1.
In the present paper, we report on the effect of pH and carbonate on the hydrolysis rate constants of N-blocked and free aminoacyl adenylate anhydrides. Whereas the hydrolysis of free aminoacyly adenylates seems principally catalyzed by OH –, the hydrolysis of the N-blocked species is also catalyzed by H +, giving this compound a U-shaped hydrolysis vs. pH curve. Furthermore, at pH's<8, carbonate has an extreme catalytic effect on the hydrolysis of free aminoacyl-AMP anhydride, but essentially no effect on the hydrolysis of N-blocked aminoacyl-AMP anhydride. Furthermore, the N-blocked aminoacyl-AMP anhydride is a very efficient generator of peptides using free glycine as acceptor. The possible significance of the observations to prebiological peptide synthesis is discussed. 相似文献
2.
Proteinase k, a seryl-protease obtained from Tritirachium album, is able to specifically hydrolyze N-blocked aminoacyl transfer ribonucleic acids (tRNAs). The blocked amino acid is released, and the tRNA molecule remains able to be recharged by its cogante amino acid. Aminoacyl-tRNAs are highly resistant to hydrolysis by the protease. This activity is not due to contamination of the protease preparation. A commercial protease from Streptomyces griseus displayed a similar activity, while trypsin, chymotrypsin, and papain unspecifically hydrolyzed all charged tRNAs tested. The characteristics of the hydrolysis performed by proteinase k closely resemble the peptidyl-tRNA hydrolase activity described in different cells as a scavenger for the peptidyl-tRNA that eventually falls from the polysomes. Out results warn about a hasty identification of any N-blocked aminoacyl-tRNA hydrolase activity in the cytoplasm as an independent peptidyl-tRNA hydrolase. 相似文献
3.
猪肝酯酶是手性合成中重要的水解酶,在猪肝酯酶的催化下,苯乙二醇环碳酸酯发生水解,生成苯乙二醇。实验围绕影响猪肝酯酶催化反应活性的4个主要因素进行了系统研究,得到了最优的酶浓度(15g/L)、pH值(8.0)、温度(25~30℃)及有机溶剂种类和浓度(二氧六环,65%v/v),为猪肝酯酶催化苯乙二醇环碳酸酯反应的进一步研究奠定了基础。 相似文献
4.
We have used a novel spectrophotometric method to study the hydrolysis of N-acetylphenylalanyl adenylate anhydride (AcPhe-AMP) and phenylalanyl-adenylate anhydride (Phe-AMP) at low concentrations (10 –5 M), 25 °C, constant buffer concentration (0.05 M), and as a function of pH. While Phe-AMP is susceptible principally to attack by OH –, with two different rates depending on whether the -amino group of the amino acid is protonated or not, the AcPhe-AMP is susceptible to acid decomposition as well. At pH's 4–8, the Phe-AMP hydrolyzes faster than the AcPhe-AMP, but at pH less than 4 or pH greater than 8, the blocked form hydrolyzes faster. Both forms are also attacked by H 2O, and at the same rate. Moreover, the hydrolysis of Phe-AMP is shown to be greatly catalyzed by carbonate, although the AcPhe-AMP is not subject to such catalysis. The rate laws for the various mechanisms and the activation energies for the hydrolyses at pH 7.1 are given. 相似文献
5.
We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp–hydroxamate–Fe 3+ complex and the formation of l-Trp–l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed. 相似文献
6.
Studies of the properties of aminoacyl derivatives of 5′-AMP are aimed at understanding the origin of the process of protein synthesis. Aminoacyl (2′,3′) esters of 5′-AMP can serve as models of the 3′-terminus of aminoacyl tRNA. We report here on the relative rates of hydrolysis of AC -D - and L -Phe AMP esters as a function of pH. At all pHs above 3, the rate constant of hydrolysis of the AC -L -Phe ester is 1.7 to 2.1 times that of AC -D -Phe ester. The D -isomer seems partially protected from hydrolysis by a stronger association with the adenine ring of the 5′-AMP. © 1993 Wiley-Liss, Inc. 相似文献
7.
N-Acetylglycyl adenylate anhydride has been shown to be readily converted in high yield to N-acetylglycyl imidazolide in the presence of excess imidazole at pH 7. The aminoacyl group can then be transferred from the imidazolide to become esters of mono- or polynucleotides. These observations suggest that histidine may be in the active site of the aminoacyl-tRNA synthetases, catalyzing the transfer of aminoacyl groups from the adenylate to tRNA. 相似文献
8.
Imidazole catalysis of phenylalanyl transfer from phenylalanine adenylate anhydride to the hydroxyl groups of homopolyribonucleotides was investigated as a chemical model of the biochemical aminoacylation of tRNA. Imidazole catalyzed transfer of phenylalanine to poly(U) increases from pH 6.5 to 7.7 and decreases above pH 7.7. At pH 7.7 approximately 10% of the phenylalanyl residues are transferred to poly(U). At pH 7.1, transfer to poly(U) was five times as great as to poly(A) and transfer to a poly(A) poly(U) double helix was negligible. At pH 7.1 approximately 45 mole percent linkages to poly(U) were monomeric phenylalanine; the remainder of the linkages were peptides of phenylalanine. The number of linkages and their lability to base and neutral hydroxylamine indicates that phenylalanine and its peptides are attached as esters to the 2' hydroxyl groups throughout poly(U) and the 2' (3') hydroxyl groups at the terminus of poly(U). These results do model the contemporary process of aminoacyl transfer to tRNA and continue to suggest that a histidine residue is in the active site of aminoacyl-tRNA-synthetases. 相似文献
9.
Abstract Peptidinol adenylates were assembled by condensing the carboxyl group of N-blocked peptides with the alkylamino group of leucinol 5′ adenosine phosphodiester. The latter was prepared as protected precursor from adenosine and leucinol by phosphite chemistry. The title compounds, which mimic aminoacyl adenylates, are designed to penetrate microorganisms via peptide permeases and to interfere with genetic translation. 相似文献
10.
Studies of crude extracts of pea seeds (Pisum sativum, var. Green feast) revealed the presence of three enzymes that hydrolyse the amide bond of aminoacyl beta-naphthylamides. They differ in their specificity towards the aminoacyl moiety; one is proline-specific, whereas the other two hydrolyse the beta-naphthylamides of primary amino acids. Of the latter, one is highly specific for hydrophobic aminoacyl residues whereas the other has a broader, somewhat complementary specificity, showing preferential hydrolysis of non-hydrophobic aminoacyl residues. These latter two aminoacyl-beta-naphthylamidases have been separated and partly characterized with regard to substrate specificity and antagonism by inhibitors. Both are true aminopeptidases, requiring the presence of a free amino group and hydrolysing the amide bonds of amino acid amides, dipeptides and oligopeptides consecutively from the N-terminal end. 相似文献
11.
Recycled paper mills produce large quantities of fibrous rejects and fines which are usually sent to landfills as solid waste. These cellulosic materials can be enzymatically hydrolyzed into sugars for the production of biofuels and biomaterials. Paper mill wastes also contain large amounts of calcium carbonate which inhibits cellulase activity. The calcium carbonate (30%, w/w) decreased 40–60% of sugar yield of unbleached softwood kraft pulp. The prime mechanisms for this are by pH variation, competitive and non-productive binding, and aggregation effect. Addition of acetic acid (pH adjustment) increased the sugar production from 19 to 22 g/L of paper mill waste fibers. Strong affinity of enzyme—calcium carbonate decreased free enzyme in solution and hindered sugar production. Electrostatic and hydrogen bond interactions are mainly possible mechanism of enzyme—calcium carbonate adsorption. The application of the nonionic surfactant Tween 80 alleviated the non-productive binding of enzyme with the higher affinity on calcium carbonate. Dissociated calcium ion also inhibited the hydrolysis by aggregation of enzyme. 相似文献
12.
In the course of the enzymatic reaction of acetoacetyl-CoA with maleate, catalyzed by CoA transferase, a transient appearance of free CoA-SH occurred. Subsequently, both free CoA and acyl-CoA decreased with time, indicating the formation of an unusual CoA derivative resistant to alkaline hydrolysis. During the chemical reaction of CoA-SH with maleic anhydride, the SH groups of CoA disappeared quickly, but not more than 30% could be accounted for as thioester. The product is unstable at neutrality and is hydrolyzed by nitroprusside reagent. Another product having an acyl bond of low reactivity, which reacts with hydroxylamine and does not undergo ammonolysis, but is susceptible to alkaline hydrolysis, slowly accumulated, accounting for about 25% of the CoA that disappeared. The main product appears to be formed by the addition of CoA-SH to the double bond of maleic anhydride. Column chromatography of the products of the chemical and the enzymatic reaction revealed two products, designated at X1 and X2, showing absorbance of the adenine moiety of CoA, containing 14C-labeled maleate and no free SH groups. Alkaline hydrolysis of X1 resulted in the recovery of CoA and in an increase of X2. The results are interpreted as indicating that maleyl-CoA readily hydrolyzes and reacts spontaneously with the SH group of free CoA to form an addition compound. The thioester of this product slowly hydrolyzes to give rise to the final product, which appears to be the thioether, a stable and metabolically inert compound. 相似文献
13.
We developed a spectrophotometric assay for peptide hydrolysis by aminopeptidases (APs). The assay enables the measurement
of free amino acids liberated by AP-catalyzed peptide hydrolysis using 4-aminoantipyrine, phenol, peroxidase, and l-amino acid oxidase. We investigated the specificity of bacterial APs [enzymes from Streptomyces griseus (SGAP), Streptomyces septatus (SSAP), and Aeromonas proteolytica (AAP)] toward peptide substrates using this assay method. Although these enzymes most efficiently cleave leucyl derivatives
among 20 aminoacyl derivatives, in peptide hydrolysis, the catalytic efficiencies of Phe-Phe hydrolysis by SGAP and SSAP exceed
that of Leu-Phe hydrolysis. Furthermore, all enzymes showed the maximum catalytic efficiencies for Phe-Phe-Phe hydrolysis.
These results indicate that the hydrolytic activities of bacterial APs are affected by the nature of the penultimate residue
or flanking moiety and the length of the peptide substrate. 相似文献
14.
Several pathovars of Pseudomonas syringae produce the phytotoxin coronatine (COR), which contains an unusual amino acid, the 1-amino-2-ethylcyclopropane carboxylic acid called coronamic acid (CMA), which is covalently linked to a polyketide-derived carboxylic acid, coronafacic acid, by an amide bond. The region of the COR biosynthetic gene cluster proposed to be responsible for CMA biosynthesis was resequenced, and errors in previously deposited cmaA sequences were corrected. These efforts allowed overproduction of P. syringae pv. glycinea PG4180 CmaA in P. syringae pv. syringae FF5 as a FLAG-tagged protein and overproduction of P. syringae pv. tomato CmaA in Escherichia coli as a His-tagged protein; both proteins were in an enzymatically active form. Sequence analysis of CmaA indicated that there were two domains, an adenylation domain (A domain) and a thiolation domain (T domain). ATP-(32)PP(i) exchange assays showed that the A domain of CmaA catalyzes the conversion of branched-chain L-amino acids and ATP into the corresponding aminoacyl-AMP derivatives, with a kinetic preference for L-allo-isoleucine. Additional experiments demonstrated that the T domain of CmaA, which is posttranslationally modified with a 4'-phosphopantetheinyl group, reacts with the AMP derivative of L-allo-isoleucine to produce an aminoacyl thiolester intermediate. This covalent species was detected by incubating CmaA with ATP and L-[G-(3)H]allo-isoleucine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. It is postulated that the L-allo-isoleucine covalently tethered to CmaA serves as the substrate for additional enzymes in the CMA biosynthetic pathway that catalyze cyclopropane ring formation, which is followed by thiolester hydrolysis, yielding free CMA. The availability of catalytically active CmaA should facilitate elucidation of the details of the subsequent steps in the formation of this novel cyclopropyl amino acid. 相似文献
15.
Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite. 相似文献
16.
Acid phosphatase purified from maize scutellum, upon acylation with succinic anhydride, still shows negative co-operativity for the hydrolysis of glucose-6-phosphate at pH 5.4. This phenomenon is abolished by glucose, for both native and succinylated enzymes, through stimulation of the initial velocities at sub-optimal substrate concentrations. However, negative co-operativity for the enzymatic hydrolysis of p-nitrophenylphosphate at pH 5.4 is suppressed only at high concentrations of glucose. Furthermore, the hydrolysis of p-nitrophenylphosphate is noncompetitively inhibited (low affinity form of the enzyme molecule) by glucose, which suggests the existence of different substrate binding sites. 相似文献
17.
Amino acids are quantitatively recovered from aminoacyl-tRNA in filter paper strips by treatment with 0.3 m ammonium carbonate. A simple apparatus is described for deaminoacylation and separation of amino acids and tRNA. The amino acids, thus recovered, can be directly analyzed by paper chromatography since ammonium carbonate is removed by lyophilization. Nonvolatile reagents such as Tris-HCl and sodium carbonate also hydrolyze aminoacyl-tRNA, but unless the products of deaminoacylation are desalted, the presence of these reagents gives rise to chromatographic artifacts such as peak broadening, appearance of multiple peaks, displacement of Rf values, etc.tRNA remaining after deaminoacylation retains only a small fraction of its original capacity to bind amino acids. Part of this loss of activity appears to be due to structural damage to tRNA in the course of washing and counting procedures and in part, due to the effect of filter paper in shifting the equilibrium of the reaction toward hydrolysis of aminoacyl ester bond. 相似文献
18.
氨基酰tRNA合成酶(aminoacyl-tRNA synthetases,aaRS)家族的经典功能是催化氨基酸与对应tRNA结合,形成氨基酰tRNA,参与蛋白质合成。aaRS在进化过程中不断增加与氨基酰化功能无关的新结构域,其亚细胞器定位也受到营养、压力信号、参与调控血管新生和炎症反应等内外部信号调控,且不同aaRS的突变导致不同人类疾病,提示aaRS具有信号传导功能,但缺少具体的生化机制。最新发现aaRS具有氨基酰转移酶活性。一种氨基酸可以被其对应的aaRS活化成氨基酰AMP,氨基酰AMP可以修饰与该aaRS相互作用蛋白质的赖氨酸,传递该氨基酸的丰度及结构信息,调控细胞信号网络。aaRS新功能的发现和研究,为解释aaRS的生理病理重要性提供新的方向。本文综述aaRS的进化及非经典功能,讨论aaRS氨基酰转移酶活性在细胞信号传导及其与疾病的相关性,也包括药物开发潜力。 相似文献
19.
Kinetic studies with substrate analogs and group-directed chemical modification agents were carried out for the purpose of identifying the enzyme-substrate interactions required for phosphonoacetaldehyde (P-Ald) binding and catalyzed hydrolysis by P-Ald hydrolase (phosphonatase). Malonic semialdehyde (Ki = 1.6 mM), phosphonoacetate (Ki = 10 mM), phosphonoethanol (Ki = 10 mM), and fluorophosphate (Ki = 20 mM) were found to be competitive inhibitors of the enzyme but not substrates. Thiophosphonoacetaldehyde and acetonyl phosphonate underwent phosphonatase-catalyzed hydrolysis but at 20-fold and 140-fold slower rates, respectively, than did P-Ald. In the presence of NaBH4, acetonyl-phosphonate inactivated phosphonatase at a rate exceeding that of its turnover. Sequence analysis of the radiolabeled tryptic peptide generated from [3-3H]acetonylphosphonate/NaBH4-treated phosphonatase revealed that Schiff base formation had occurred with the catalytic lysine. From the Vm/Km and Vm pH profiles for phosphonatase-catalyzed P-Ald hydrolysis, an optimal pH range of 6-8 was defined for substrate binding and catalysis. The pH dependence of inactivation by acetylation of the active site lysine with acetic anhydride and 2,4-dinitrophenyl acetate evidenced protonation of the active site lysine residue as the cause for activity loss below pH 6. The pH dependence of inactivation of an active site cysteine residue with methyl methanethiol-sulfonate indicated that deprotonation of this residue may be the cause for the loss of enzyme activity above pH 8. 相似文献
20.
The kinetic alpha-deuterium isotope effect on Vmax/Km for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 +/- 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic alpha-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of Vmax/Km change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same. Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent. In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic alpha-deuterium isotope effect of unity. Thus, covalent bond-changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several precedents. 相似文献
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