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Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9') and of the serpin molecule in general. The "hinge" region of the reactive center loop, including residues P10-P14, shows facile movement in and out of beta-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the alpha1-antichymotrypsin.alpha-chymotrypsin encounter complex, E.I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of alpha1-antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates, EIa, EIb, and EIc, between E.I and E*I* and permit informed speculation regarding the nature of these intermediates. Partitioning between inhibitor and substrate pathways occurs late in the process of E*I* formation, most likely from a species occurring between EIc and E*I*. 相似文献
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R Gandolfi F Marinelli E Ragg D Romano F Molinari 《Bioorganic & medicinal chemistry letters》2012,22(16):5283-5287
The chemoenzymatic deacylation of ramoplanin A2 is described for the first time: ramoplanin A2 was Boc-protected and hydrogenated to Boc-protected tetrahydroramoplanin, which was subsequently deacylated using an acylase from Actinoplanes utahensis NRRL 12052. The chemoenzymatic process proceeded with 80% overall yield, which favourably compares with the previously described chemical deacylation. 相似文献
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Specificity in deacylation of acyl-alpha-chymotrypsins 总被引:3,自引:0,他引:3
D W Ingles J R Knowles J A Tomlinson 《Biochemical and biophysical research communications》1966,23(5):619-625
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Glynn P 《Biochimica et biophysica acta》2005,1736(2):87-93
Certain organophosphates react with the active site serine residue of neuropathy target esterase (NTE) and cause axonal degeneration and paralysis. Cloning of NTE revealed the presence of homologues in eukaryotes from yeast to man and that the protein has both a catalytic and a regulatory domain. The latter contains sequences similar to the regulatory subunit of protein kinase A, suggesting that NTE may bind cyclic AMP. NTE is tethered via an amino-terminal transmembrane segment to the cytoplasmic face of the endoplasmic reticulum. Unlike wild-type yeast, mutants lacking NTE activity cannot deacylate CDP-choline pathway-synthesized phosphatidylcholine (PtdCho) to glycerophosphocholine (GroPCho) and fatty acids. In cultured mammalian cells, GroPCho levels rise and fall, respectively, in response to experimental over-expression, and inhibition, of NTE. A complex of PtdCho and Sec14p, a yeast phospholipid-binding protein, both inhibits the rate-limiting step in PtdCho synthesis and enhances deacylation of PtdCho by NTE. While yeast can maintain PtdCho homeostasis in the absence of NTE, certain post-mitotic metazoan cells may not be able to, and some NTE-null animals have deleterious phenotypes. NTE is not required for cell division in the early mammalian embryo or in larval and pupal forms of Drosophila, but is essential for placenta formation and survival of neurons in the adult. In vertebrates, the relative importance of NTE and calcium-independent phospholipase A2 for homeostatic PtdCho deacylation in particular cell types, possible interactions of NTE with Sec14p homologues and cyclic AMP, and whether deranged phospholipid metabolism underlies organophosphate-induced neuropathy are areas which require further investigation. 相似文献
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An extensive comparison of deacylation rates of mono- and disubstituted benzoyl-α-chymotrypsins indicates that no steric effects on rate or apparent pKa of deacylation are detectable within this series. Some anomalous effects on deacylation rate appear to be associated with fluoro- and nitro-substituents in particular positions on the ring and may be attributable to specific interactions at the enzyme active site. The extensive series of structurally similar acyl-enzymes prepared has allowed a thorough analysis of the effect of acyl group pKa on the apparent pKa of deacylation. The data indicates that polar effects on the apparent pKa are probably negligible. Rho for the deacylation reaction is in good agreement with model reactions for an imidazole general base-catalyzed model reaction. 相似文献
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The method of chemical aggregation of enzymes has the advantage of yielding an immobilized enzyme preparation wherein reactor volume can be significantly reduced because of the absence of an inert carrier. A coaggregate of trypsin and chymotrypsin formed by extensive cross-linking with glutaraldehyde is described. A significant property of this aggregate is the reduced autolysis of the trypsin component of the coaggregate. 相似文献
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A kinetic study of procine chymotrypsin A-pi revealed two characteristic properties of this type of chymotrypsin: 1. Porcine chymotrypsin A-pi, like bovine chymotrypsin B-pi does not bind proflavin, which is a competitive inhibitor of bovine trypsin and chymotrypsin A-alpha. 2. The pH profiles of the steady-state parameters show the two usual important pK's. The basic one, pK2 = 9.6, affects both Km and kcat/Km and probably controls the binding conformation of chymotrypsin. The acidic one, pK1 = 5.7, affects kcat and kcat/Km and plays a role in the catalytic process. The value of pK1 is unusually low. 相似文献
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Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced. 相似文献
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Patton-Vogt J 《Biochimica et biophysica acta》2007,1771(3):337-342
Phospholipid deacylation results in the formation of glycerophosphodiesters and free fatty acids. In Saccharomyces cerevisiae, four gene products with phospholipase B (deacylating) activity have been characterized (PLB1, PLB2, PLB3, NTE1), and those activities account for most, if not all, of the glycerophosphodiester production observed to date. The glycerophosphodiesters themselves are hydrolyzed into glycerol-3-phosphate and the corresponding alcohol by glycerophosphodiester phosphodiesterases. Although only one glycerophosphodiester phosphodiesterase-encoding gene (GDE1) has been characterized in S. cerevisiae, others certainly exist. Both internal and external glycerophosphodiesters (primarily glycerophosphocholine and glycerophosphoinositol) are formed as a result of phospholipid turnover in S. cerevisiae. A permease encoded by the GIT1 gene imports extracellular glycerophosphodiesters across the plasma membrane, where their hydrolytic products can provide crucial nutrients such as inositol, choline, and phosphate to the cell. The importance of this metabolic pathway in various aspects of S. cerevisiae cell physiology is being explored. 相似文献
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Masayuki Nakagawa Toshiie Nakamura Kazuo Tomita 《Bioscience, biotechnology, and biochemistry》2013,77(11):2205-2208
Photolysis of 3-hydroxy-5-phenylisoxazole in methanol with a low-pressure mercury lamp afforded 5-phenyl-4-oxazolin-2-one, together with small amounts of benzoic acid and benzoylacetamide. Similarly, 3-hydroxy-5-methylisoxazole in distilled water afforded 5-methyl-4-oxazolin-2-one as the major product. Both isoxazoles were stable in sunlight for up to 20 days. 相似文献
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