Acetate-activating enzyme

D-Glucosamine 6-phosphate can be readily acetylated by a AT-acetylase obtained from a preparation of yeast hexokinase. The resulting iV-acetyl-D-glucosamine 6-phosphate is identified by its Morgan-Elson reaction. The acetyl-coenzyme A which appears to be required for this reaction may be generated by acetate, adenosine-5-triphosphoric acid, and coenzyme A (in the presence of an acetate-activating enzyme). 

Fatty Acid Activation. The activation of fatty acids [reaction (1)] is catalyzed by enzymes that are specific for various chain lengths. The first of these, the acetate-activating enzyme, acetic thiokinase, has... 

Chohnesterase-inhibiting pesticides (e g., organophosphate and carbamate pesticides) are detected by dipping the developed chromatogram in a solution of the enzyme chohnesterase followed by incubation for a short period. Then the plate is dipped in a substrate solution, e.g., 1-naphthyl acetate/fast blue salt B. In the presence of the active enzyme, 1-naphthyl acetate is hydrolyzed to 1-naphthol and acetic acid, and the 1-naphthol is coupled with fast blue salt B to form a violet-blue azo dye. The enzyme is inhibited by the pesticide zones, so the enzyme-substrate reaction does not occur pesticides are, therefore, detected as colorless zones on a violet-blue background [36]. 

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

Manganese acts as a cofactor of mevalonate kinase and farnesyl pyrophosphate synthetase. Mevalonate kinase and possibly one other manganese-activated enzyme are necessary for the formation of mevalonate from acetate (3). Farnesyl pyrophosphate synthetase acts to add one 5-carbon unit to geranyl pyrophosphate to make farnesyl pyrophosphate (4) (Figure 1). 

Figure 11.2 Kinetic plots (initial rate versus sucrose concentration) of fructosyltransferase from A aculeatus. Transfer activity ( ) and hydrolytic activity (o). Reactions were carried out in 0.2 M sodium acetate buffer (pH 5.5) at 60°C. Kinetic parameters were calculated estimating a molecular mass of 135 kDa for the active enzyme. Adapted from Ref [33].

At least three acyl-CoA synthases, each specific for a particular size of fatty acid, exist acetyl-CoA synthase acts on acetate and other low-molecular-weight carboxylic acids, medium-chain acyl-CoA synthase on fatty acids with 4-11 carbon atoms, and acyl-CoA synthase on fatty acids with 6-20 carbon atoms. The activity of acetyl-CoA synthase in muscle is restricted to the mitochondrial matrix. Medium-chain acyl-CoA synthase occurs only in liver mitochondria, where medium-chain fatty acids obtained from digestion of dietary triacylglycerols and transported by the portal blood are metabolized. Acyl-CoA synthase, the major activating enzyme, occurs on the outer mitochondrial membrane surface and in endoplasmic reticulum. The overall reaction of activation is as follows ... 

It was found that the enzyme is specific for (/ )-ATPaS but does not react with (S)-ATPaS. As shown in Scheme 43, when (/ )-ATPatS and l70-acetate are used as substrates, the 170 from acetate will be incorporated into the pro-5 position of AMPS if the reaction proceeds with retention of configuration or into the pro-/ position if inversion occurs. To determine the configuration of the 170-labeled AMPS (compound type 4), it is converted to (S)-ATPoS by stereospecific phosphorylation at the pro-/ oxygen catalyzed by adenylate kinase, followed by a second phosphorylation catalyzed by pyruvate kinase (144,145). By such a conversion, 170 should be incorporated into the nonbridging position of (5)-ATPaS if the step of acetate activation proceeds with retention of configuration. On the other hand, 170 should be located at the P—O—P bridging... 

Type 2 copper centers are not uniform in ligand or ligand stereochemistries. One common feature is, however, that in the active enzyme, one coordination site is always free to bind oxygen. The most common ligand in type 2 copper centers is histidine. Tyrosine (often modified), methionine, and cysteine occur as well. There are three histidines and a modified tyrosine in amine oxidase and lysyl oxidase [28]. In diamine oxidase, two of the histidine residues have probably been replaced by cysteines [29]. In galactose oxidase, the copper ion is coordinated by two tyrosines, two histidines and an acetate ion [30]. Dopamine-/J-hydroxylase contains two differently coordinated copper ions per functional unit. One is coordinated by three histidines and a methionine and the other by two histidines and another, yet unknown, ligand [ 31 ]. Last but not least, the type 2 copper ion in Cu,Zn-superoxide dismutase is coordinated by four histidine residues, one of which connects the copper ion to the zinc ion, the second metal ion in the active site of the enzyme [32,33] (Fig. 6). 

The XANES of the Mn catalase provided the first definitive proof that this enzyme cycles between the MnJ and Mn n oxidation levels (137, 352) The extent of catalase activity correlated with the proportion of MnJ1 or Mn enzyme however, samples with Mnlv quantitatively showed reduced activity. The EXAFS of the Mn catalases have been less informative because of the Mn-Mn separations in the reduced, active enzymes (135). Nevertheless, EXAFS of the superoxi-dized enzyme demonstrated that the Mn,uMnlv enzyme has a Mn-Mn separation of —2.7 A, which is consistent with a di-yu.2-oxo core (135). Subsequent spectroscopic analysis confirmed that a diamond core with a bridging syn,syn acetate formed the enzyme active site (9). 

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