Deacylase activity

ECB deacylase is produced naturally by Actinoplanes utahensis. However, very little (—0.2%) deacylase activity was detected in the culture filtrate of Actinoplanes utahensis. Less than 5% of the cell-associated deacylase activity was released by incubation of the cells at 0.01 M KH2P04, pH 6, for 1 day. A simple salt treatment with 0.8 M KC1 resulted in 60-80% recovery of soluble deacylase at a high specific activity [25], This salt-induced solubilization suggests that the deacylase is loosely bound to the membrane of A. utahensis and can be released by disruption of ionic interactions [26]. The solubilized enzyme was stable and purified to apparent homogeneity by a four-step conventional procedure [25]. 

Purified MCBP was incubated with [l-14C]myristoyl-CoA in separate tubes and aliquots were removed at selected times. At time 10 min, cytosolic fraction was added to one tube and buffer was added to the other. Aliquots were removed at selected times up to 120 min. The vial which did not contain added cytosol exhibited the formation of a protein-myristoyl-CoA complex, while the tube which contained added cytosol exhibited deacylation (Figure 17.4B). These results suggest that the cytosolic fraction may contain thioesterases/proteinases which could modulate the acylation reaction in vivo (Raju and Sharma 1997). The absence of acyl-complex formation in the cytosol could be due to the presence of either esterases or pro-teinases. It has been reported that porcine phospholipase A contained thioesterase and deacylase activities (Nocito et al. 1996). 

Nocito, M., Roy, G., Villar, L. M., Palacios, C., Serrano, A., Alvarez-Cermeno, J. C., and Gonzalez-Porque, P. 1996. Thioesterase and protein deacylase activities of porcine pancreatic phospholipase A2. Biochim. Biophys. Acta 1299 17-22. 

It was suggested that deacylase activity exists at least for the amide substituent hydrolysis of the 2 -0-acyl substituent was not studied b. 

A single patient with multiple congenital malformations was reported with isolated 3-hydroxyisobutyryl-CoA deacylase deficiency [18]. Intact cell oxidation of [ C]-valine revealed decreased pathway fimction, and direct analysis in extracts of fibroblasts derived from the patient revealed deficient deacylase activity. The urine contained increased amounts of unusual sulfhydryl adducts, S-(2-carboxypropyl)cysteine and S-(2-carboxypropyl-cysteamine), believed to represent the cysteine and cysteamine conjugates of methacrylyl-CoA. The latter, a highly reactive species, was postulated to be the pathologic intermediate leading to physical malformations. 

Spencer and Maung (277) isolated a protein from P. chrysogenum which, after purification, appeared to be homogeneous and possessed acyltransferase activity, acylase and deacylase activity, and phenylacetyl-CoA hydrolase activity. 

Three compounds acetoacetate, P-hydroxybutyrate, and acetone, are known as ketone bodies. They are suboxidized metabolic intermediates, chiefly those of fatty acids and of the carbon skeletons of the so-called ketogenic amino acids (leucine, isoleucine, lysine, phenylalanine, tyrosine, and tryptophan). The ketone body production, or ketogenesis, is effected in the hepatic mitochondria (in other tissues, ketogenesis is inoperative). Two pathways are possible for ketogenesis. The more active of the two is the hydroxymethyl glutarate cycle which is named after the key intermediate involved in this cycle. The other one is the deacylase cycle. In activity, this cycle is inferior to the former one. Acetyl-CoA is the starting compound for the biosynthesis of ketone bodies. 

For example, in the case of ECB deacylase the initial investigations showed that the enzyme was most active at high pH and temperature (e.g., pH 8, 70°C), but that the product (ECB nucleus) degraded rapidly under these conditions. One solution was to perform the bioconversion at lower pH and temperature, where the product would be stable, and accept suboptimal enzyme performance (i.e., slower reaction time or more enzyme usage). 

Rutten, L., Mannie, J.P., Stead, C.M., Raetz, C.R., Reynolds, C.M., Bonvin, A.M., Tommassen, J.P., Egmond, M.R., Trent, M.S., Gros, P. Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium. Proc Natl Acad Sci USA 106 (2009)... 

These enzymatic activities include three acyltransferases (HtrBl, HtrB2, PagP) and a deacylase (PagL). Depending on the specific PA isolate background, these activities can be classified as inducible (lipid A structures only observed under specific growth conditions that induce modification in these isolates), constitutive (lipid A structures always observed under any growth condition in these... 

Paclitaxel and related compounds have also been found in various Taxus species in addition to the Pacific yew, occurring in roots, stems, wood, and needles as well as bark. Yew extracts contain a complex mixture of taxanes, with paclitaxel usually constituting less than 20% of the total taxanes. Isolation of paclitaxel from these mixtures is a difficult purification problem and contributed to the slow development of this compound as a drug. The most valuable material in this mixture for semisynthesis is 10-deacetylbaccatin-III. Microbial strains were isolated from soil samples containing C-13 deacylase and C-lO-deacetylase enzyme activities that are able to convert mixtures of taxanes to 10-deacetylbaccatin-III, thereby increasing the amount and ease of isolation of this precursor for semisynthesis (Scheme 17.14). Treatment of ethanol extracts, prepared either from whole plants of a variety of renewable yew cultivars or from material derived from the bark of... 

Tervo et al. [155] described a successful virtual screening experiment for the class III histone deacylase sirtuin type 2 (SIRT2). Using two databases of commercially available compounds plus a receptor-based pharmacophore query, 4 out of 11 tested molecules showed weak to moderate inhibitory activity. The two most active inhibitors vdth IC50 values of 51 and 91 pM had new scaffolds (Figure 12.14b). 

One especially interesting method for resolving amino acids is based on the use of enzymes called deacylases. These enzymes catalyze the hydrolysis of N-acylamino acids in living organisms. Since the active site of the enzyme is chiral, it hydrolyzes only A/ acylamino acids of the l configuration. When it is exposed to a racemic mixture of A/ acylamino acids, only the derivative of the L-amino acid is affected and the products, as a result, are separated easily ... 

Two observations suggest that ketosis in diabetes results from the combination of the accelerated hydroxymethylglutarate shunt and the increased rate of deacylation. Mitochondrial deacylation is the major pathway for converting acetoacetyl CoA to acetoacetic acid in both normal and alloxan-diabetic livers, and a marked increase in activity of acetoacetyl CoA deacylase and a moderate increase in the hydroxymethylglutarate cleavage enzymes is observed in alloxan-diabetic rats. 

Acetoacetate Metabolism. An active deacylase in liver is responsible for the formation of free acetoacetate from its CoA derivative. The j8-hydroxybutyric dehydrogenase mentioned above and a decarboxylase are capable of converting acetoacetate into the other ketone bodies, /3-hydroxybutyrate, and acetone. liver does not contain a mechanism for activating acetoacetate. Heart muscle has been found to contain a specific thiophorase that forms acetoacetyl CoA at the expense of suc-cinyl CoA. Acetoacetate is thus used by peripheral tissues by activation through transfer, then reaction with either the enzymes of fatty acid synthesis or jS-ketothiolase and the enzymes that use acetyl CoA. 

One especially interesting method for resolving amino acids is based on the nse of enzymes called deacylases. These enzymes catalyze the hydrolysis of N-acylamino acids in living organisms. Since the active site of the enzyme is chiral, it hydrolyzes only... 

Fatty acid oxidation can be terminated in either of two ways. Acetoacetyl CoA can either be cleaved to two molecules of acetyl CoA which condense with oxalacetate to form citrate, or it can be deacylated to acetoacetate by a deacylase specific for d-keto butyryl CoA. - In kidney and heart muscle there is no accumulation of acetoacetate, whereas in liver acetoacetate is formed in preference to citrate. The non-accumulation of acetoacetate in tissues other than liver probably is referable to the following circumstances. All tissues but liver contain activating enzymes which catalyze the conversion of acetoacetate to acetoacetyl CoA. Thus any acetoacetate formed by deacylation is thrust back as it were into the metabolic wheel. In liver deacylation is not opposed by this reactivation of acetoacetate. Hence acetoacetate accumulates only in liver. 

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