Transfer RNA (tRNA) is an essential component of the cell's translation apparatus. These RNA strands contain the anticodon for a given amino acid, and when "charged" with that amino acid are termed aminoacyl-tRNA. Aminoacylation, which occurs exclusively at one of the 3'-terminal hydroxyl groups of tRNA, is catalyzed by a family of enzymes called aminoacyl-tRNA synthetases (ARSs). In a primitive translation system, before the advent of sophisticated protein-based enzymes, this chemical event could conceivably have been catalyzed solely by RNA enzymes. Given the evolutionary implications, our group attempted in vitro selection of artificial ARS-like ribozymes, successfully uncovering a functional ribozyme (r24) from an RNA pool of random sequences attached to the 5'-leader region of tRNA. This ribozyme preferentially charges aromatic amino acids (such as phenylalanine) activated with cyanomethyl ester (CME) onto specific kinds of tRNA. During the course of our studies, we became interested in developing a versatile, rather than a specific, aminoacylation catalyst. Such a ribozyme could facilitate the preparation of intentionally misacylated tRNAs and thus serve a convenient tool for manipulating the genetic code. On the basis of biochemical studies of r24, we constructed a truncated version of r24 (r24mini) that was 57 nucleotides long. This r24mini was then further shortened to 45 nucleotides. This ribozyme could charge various tRNAs through very simple three-base-pair interactions between the ribozyme's 3'-end and the tRNA's 3'-end. We termed this ribozyme a "flexizyme" (Fx3 for this particular construct) owing to its flexibility in addressing tRNAs. To devise an even more flexible tool for tRNA acylation, we attempted to eliminate the amino acid specificity from Fx3. This attempt yielded an Fx3 variant, termed dFx, which accepts amino acid substrates having 3,5-dinitrobenzyl ester instead of CME as a leaving group. Similar selection attempts with the original phenylalanine-CME and a substrate activated by (2-aminoethyl)amidocarboxybenzyl thioester yielded the variants eFx and aFx (e and a denote enhanced and amino, respectively). In this Account, we describe the history and development of these flexizymes and their appropriate substrates, which provide a versatile and easy-to-use tRNA acylation system. Their use permits the synthesis of a wide array of acyl-tRNAs charged with artificial amino and hydroxy acids. In parallel to these efforts, we initiated a crystallization study of Fx3 covalently conjugated to a microhelix RNA, which is an analogue of tRNA. The X-ray crystal structure, solved as a co-complex with phenylalanine ethyl ester and U1A-binding protein, revealed the structural basis of this enzyme. Most importantly, many biochemical observations were consistent with the crystal structure. Along with the predicted three regular-helix regions, however, the flexizyme has a unique irregular helix that was unexpected. This irregular helix constitutes a recognition pocket for the aromatic ring of the amino acid side chain and precisely brings the carbonyl group to the 3'-hydroxyl group of the tRNA 3'-end. This study has clearly defined the molecular interactions between Fx3, tRNA, and the amino acid substrate, revealing the fundamental basis of this unique catalytic system. 相似文献
We have investigated the possibility that the nonrandom association of fatty acids in rat milk triglycerides results from specificity of the acyl transferases in the glycerolphosphate pathway. Subcellular fractionation of lactating rat mammary gland revealed that the microsomal fraction was the most active in acylation of 3-sn-[U-14C] glycerolphosphate with various acyl-CoA's. The major products were diacylglycerolphosphate and diglyceride; no monoacylglycerolphosphate was detected. Maximum rate of acylation occurred at or below the critical micelle concentration for each acyl-CoA, indicating that only the monomeric substrate molecules were acceptable by the enzyme system. The observed acyl specificity, 16∶0>18∶0≏14∶0>12∶0>10∶0>8∶0 is consistent with the concept that, in general, milk triglycerides are synthesized by insertion of a short or medium chain fatty acid into a long chain diglyceride. 相似文献
The exhibited geometry of catalytic substrates can have a significant influence on the chemical activity and efficiency. Controlling their geometry can be challenging using the traditional techniques. In this work, we propose new and novel catalytic substrates with architected and controllable topologies based on the minimal surfaces framework. A novel design approach and an additive manufacturing (AM) technique were proposed to manufacture the catalytic substrates using ceramic materials. After 3D printing, their mechanical and flow properties were investigated experimentally. An elastic-plastic-damage coupled model was employed to investigate the underlying deformation mechanism of the investigated substrates. Results showed that the CLP substrate exhibited the highest mechanical properties as well as the least pressure drop among the tested substrates. Also, numerical simulations showed that the strut-based substrates exhibit stress localization which leads to faster failure, while stress is distributed more homogeneously in the sheet-based substrates. While the model showed to have a good agreement in the experimental and simulation stress-strain responses, the damage mechanism was not fully captured by the numerical simulations. This was attributed mainly to the process-induced defects in the form of microcracks and microvoids that can alter the nature of deformation and damage. 相似文献
The substrate specificity of rat brain neurolysin was rapidly modified by semirational mutagenesis coupled with a yeast molecular display system. Neurolysin mainly recognizes substrates with sequential six residues close to the scissile bond in polypeptides, cleaving a peptide bond in the center position of the six residues. To alter the recognition of the P2' amino acid of substrates by neurolysin, six residues of neurolysin, Asp467, Arg470, Glu510, Tyr606, Tyr610 and Tyr611, which might be involved in the formation of the neurolysin S2' subsite, were individually and comprehensively substituted. The protein libraries of mutant neurolysins comprising 120 species were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) and MMP-3-specific substrates, which consisted of similar amino acids, except for alanine (for MMPs-2/9) or glutamic acid (for MMP-3) at the P2' amino acid position. Among mutant neurolysins, the Y610L mutant neurolysin exhibited a marked change in substrate specificity. Steady-state kinetic analysis of the purified Y610L mutant neurolysin revealed that the binding efficiency toward the MMP-3-specific substrate was about 3-fold higher than that toward the MMP-2/9-specific substrate. These results indicate that Tyr610 of neurolysin is the important residue to recognize the P2' amino acid of substrates. 相似文献
4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H), a flavin-dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational redesign to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged with residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2-hydroxycarbazole. Reactions were followed by an adapted Fe(III)-catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of nonaromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole-cell biocatalysts) and in vitro (three-enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of byproducts. 相似文献
The importance of Glu87 and Trp89 in the lid of Humicola lanuginosalipase for the hydrolytic activity at the water/lipid interfacewas investigated by site-directed mutagenesis. It was foundthat the effect on the hydrolytic activity upon the replacementof Trp89 with Phe, Leu, Gly or Glu was substrate dependent TheTrp89 mutants displayed an altered chain length specificitytowards triglycerides, with a higher relative activity towardstriacetin and trioctanoin compared with tributyrin. Trp89 wasshown to be lessimportant in the hydrolysis of vinyl esterscompared with ethylesters and triglycerides. An exclusive effecton the acylation reaction rate by the mutation of Trp89 wasconsistent with the data. It is suggested that Trp89 is importantin the process of binding the acyl chain of thesubstrate intothe activesite for optimal acylation reaction rate. The Trp89Phemutation resulted in an increased hydrolytic activity towards2-alkylalkanoic acid esters. This is suggested to be due toreduction of unfavourable van der Waals contacts between Trp89and the 2-substituent of the substrate. Thus, in contrast tonatural substrates, Trp89 has a negative impact on the catalyticefficiencywhen substrates with bulky acyl chains are used. Incontrast to the Trp89 mutations, the effect on the hydrolyticactivity of the Glu87Ala mutation was almost substrate independent,3570% activity of wild-type lipase. Areduction of boththe acylation and deacylation reaction was consistent with thedata. 相似文献
Acyl-CoA synthetase activity with various long chain fatty acid substrates was measured in microsomes from rat testes, isolated
spermatids and testes of hypophysectomized adult rats, using reversed-phase high performance liquid chromatography (HPLC).
The spectrophotometric HPLC method produced results comparable to those of parallel radiometric assays and was highly specific
for acyl-CoA products. At optimal pH and cofactor concentrations, specific activity from whole testis was similar for 18∶1,
20∶4 and 22∶5 but somewhat lower for 16∶0 over the substrate range 0.01–3.2 mM. Activity from spermatids or from testes of
hypophysectomized rats was much lower with 22∶5 than with 18∶1 or 20∶4, whereas activities with 18∶1 and 20∶4 were similar
at all substrate concentrations. All substrates exhibited Michaelis-Menten type saturation kinetics and linear Lineweaver-Burke
plots at lower substrate concentrations but inhibited activity at higher concentrations. Apparent values of KM for 16∶0, 18∶1 and 20∶4 were more than twice that of 22∶5, whereas both observed and calculated maximum velocities were similar
for the four fatty acids. Differences in pseudokinetic parameters and differential expression of the testicular acyl-CoA synthetase
activities with different fatty acids suggest the presence of multiple enzymes, at least one of which may be hormonally regulated. 相似文献
The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A- to P-site passage of the tRNA 3′ terminus during protein synthesis is performed by a rotary motion, synchronized with the overall tRNA/mRNA sideways movement, and guided by the PTC. This rotary motion leads to suitable stereochemistry for peptide bond formation as well as for substrate-mediated catalysis. Analysis of the substrate binding modes to ribosomes led to the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, potentially in a similar manner to the modern PTC. Later in evolution, a mechanism, enabling some type of decoding genetic control triggered the emergence of the small ribosomal subunit or part of it. This seems to be the result of the appearance of reaction products that could have evolved after polypeptides capable of enzymatic function were generated sporadically, while an ancient stable RNA fold was converted into an old version of a tRNA molecule. Since in the contemporary ribosome, the symmetry relates only to the backbone fold and nucleotide orientations but not nucleotide sequences, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC may have evolved by gene fusion or gene duplication. 相似文献
In order to characterize the several isoenzymes of soybeans, they were examined with respect to the effect of the polar nature
of the substrate. In general, lipoxygenase-1 was most active when presented with charged substrates such as the anionic form
of linoleic acid or of potassium linoleyl sulfate, whereas lipoxygenase-2 and-3 preferred nonpolar substrates such as unionized
linoleic acid, methyl linoleate, linoleyl methane sulfonate, 10,13-nonadecadieneamine, or linoleyl acetate. Linoleyl sulfate,
which has been advanced as an excellent readily soluble substrate for lipoxygenase, was indeed the best substrate found for
lipoxygenase-1. Lipoxygenase-2 and-3 were, by contrast, totally inactive against this substrate. The favorable response of
linnoleic acid to lipoxygenase-2 and-3 at pH 6.8 was ascribed to the anomalously high pKa value of linoleic acid compared to that of short chain carboxylic acids. The pH-activity profile obtained with lipoxygenase
acting on linoleyl sulfate (which was charged at all pH values examined) was shifted to lower pH values compared to the linoleic
acid activity profile. The effect of changing from the charged to the uncharged substrate, when tested against lipoxygenase-1,
was to increase the Km by an order of magnitude. 相似文献
Abstract The reaction of chlorine dioxide with different types of lignin model compounds was investigated in order to compare the kinetics and to evaluate the amount of oxidant consumed by the different substrates. Complete reaction of lignin model compounds was observed at ClO2‐to‐substrate molar ratios of 0.9–1.2, which corresponds to an electron transfer varying between 5–6 equivalents per mole of substrate. Muconic acid derivatives also fully reacted, at a ClO2‐to‐substrate molar ratio of 1.2, with the oxidant consumption being about 4 equivalents per mole of substrate. The reaction of mixtures of phenolic, non‐phenolic, and muconic acid type substrates showed that the reaction rates of non‐phenolic and muconic acid type substrates were rather similar. This study suggests that further reaction between ClO2 and the primary lignin oxidation products, such as muconic acid type structures could be the cause of overconsumption of oxidant in a D stage. 相似文献
This work describes acylation reactions facilitated by a type of heterocycle‐based acyl transfer agent, 2‐acyloxypyridazinone. Reactions of 2‐acyloxypyridazinone with carboxylic acids yield mixed carbonic anhydride intermediates, which are reactive and could be coupled with a wide range of substrates including acids, amines, alcohols, and thiols. The wide substrate scope, ease of operation (no additive or catalyst), storage and handling stability, and atom‐efficiency from recycling the heterocycle carrier make the reported acylating agent attractive for acylation‐based coupling reactions.
To observe how the stereospecific distribution of acyl groups in triglycerides is affected by the composition of fatty acids
available for esterification, the oleaginous yeastApiotrichum curvatum was grown on various binary mixture of palmitic, stearic, oleic and linoleic acids as carbon sources, and the yeast triglycerides
were analyzed. When oleic acid-linoleic acid mixtures in various ratios were used as substrates, the yeast grew well, and
the composition of the intracellular triglycerides reflected the substrate composition, but more linoleate than oleate was
deposited in the triglycerides. Oleate was favored over linoleate at thesn-2 position of the glycerol. With substrates containing palmitic and stearic acids, the yeast accumulated less oil, and incorporation
of stearic acid into the triglycerides also was very limited. When mixtures of palmitic acid-oleic acid and palmitic acid-linoleic
acid were used as substrates, the yeast triglyceride composition did not reflect that of the substrate, and the accumulation
in the yeast of the unsaturated acid in the substrate was favored. Possibly, the yeast had more limited access to solid than
to liquid substrates. For oleic acid-linoleic acid substrates, when the percentages of oleate and linoleate at the three glycerol
positions were plottedvs. the percentage of these acyl groups in the total triglyceride, apparent linear relations were observed for most of the range,
and the sums of the intercepts and slopes of the three lines for each acyl group were 0 and 3, respectively. Two mathematical
models of triglyceride assembly are proposed, both of which fit the experimental data. One model assumes that for a certain
proportion of the glycerol molecules, the acyl composition of the threesn positions is rigidly controlled independently of the substrate concentration. The other assumes that the various acyl groups
are distributed on the threesn positions of glycerol with different affinities. 相似文献
The ethylaluminium dichloride induced Friedel- Crafts acylation of unsaturated fatty compounds such as oleic acid ( 1a ), methyl oleate ( 1b ) and 10-undecenoic acid ( 9b ) and furthermore of 1-octene ( 9a ) with α,β-unsaturated acyl chlorides e.g. crotonic acid chloride ( 2a ) and acrylic acid chloride ( 2b ) gave the corresponding allyl vinyl ketones. Nazarov cyclizations of the acylation products 3a/4a, 3b/4b, 10a and 10b afforded the alkyl substituted 2-cyclopentenones 5a/6a, 5b/6b, 11a/12a and 11b/12b . Catalytic hydrogenation of 5b/6b and 11b/12b gave the respective saturated cyclic products 7b/8b and 13b/14b as diastereomeric mixtures. 相似文献