Enzyme acylation

The transport is accomplished with the participation of carnitine, which takes up the acyl from acyl-CoA on the outer membrane side. Acylcamitine assisted by carnitine translocase diffuses to the inner side of the membrane to give its acyl to the CoA located in the matrix. The process of reversible acyl transfer between CoA and carnitine on the outer and inner sides of the membrane is effected by the enzyme acyl-CoA-camitine transferase. 

There are two classes of synthetase, each with 10 members. The amino acid sequences of these two classes have regions which are identical for all enzymes of the particular class (Eriani, 1990). The class 1 synthetases acylate the tRNA at the 2 -hydroxyl of the terminal adenosine, while the class 2 enzymes acylate predominantly at the 3 -function of the ribose. 

Mahmoudian, M., Eaddy, J. and Dawson, M., Enzymic acylation of 506U78 (2-amino-9-/ -D-arabinofuranosyl-6-methoxy-9JS-purine), a powerful new anti-leukaemic agent. Biotechnol. Appl. Biochem., 1999, 29, 229-233. 

The initial oxidation reaction is unusual since it uses molecular oxygen. It is catalysed by the enzyme acyl-CoA oxidase. 

An enzymatic reaction intermediate formed by phospho-ryl transfer to a carboxyl group on an enzyme. Acyl-phosphates are structurally analogous to acid anhydrides (R—CO —O —CO—R ), and they are thermodynamically less stable than either of the two phosphoanhydride bonds in ATP. This is evident by the fact that the acetate kinase reaction (ADP + acetyl-phosphate = ATP + acetate) favors ATP formation with an equilibrium constant of about 3,000. Acetyl-phosphate can be chemically synthesized by reacting orthophosphate with acetic anhydride. 

SHIKIMATE DEHYDROGENASE SULFITE REDUCTASE VALINE DEHYDROGENASE ZEATIN aS-TRANS ISOMERASE NADPH DEHYDROGENASE NAD(P)H DEHYDROGENASE (QUINONE) NADPH-dependent enzymes, ACYL-ACYL-CARRIER-RROTEIN -DESA-TURASE... 

Figure 17.19 Rates of hydrolysis of two families of esters by a hydrolase, chymotrypsin. The esters of N-acetyl-L-phenylalanine exhibit very similar rates because the process in each case is limited by the same enzyme deacylation reaction (Zerner et al., 1964). The esters of N-benzoyl glycine exhibit rates varying by more than a factor of 3 because their hydrolyses are mostly limited by the initial enzyme acylation step (Epand and Wilson, 1963).

The formation of mixed enzyme-CoA or enzyme-acyl carrier protein disulfides was supported by the fact that the activation by these compounds was readily reversed by treatment with glutathione or cysteine (49). Preliminary experiments (50) with radioactive CoA or by titration of sulfhydryl groups in the protein suggest that maximum activation is associated with the incorporation of 4 equivalents of CoA per mole of protein (one per subunit). 

We now explore the remarkable process by which a long-chain saturated fatty acid is converted into two-carbon units (acetate), which can be oxidized to C02 and H20 via the tricarboxylic acid cycle and the electron-transport chain. Fatty acids that enter cells are activated to their CoA derivatives by the enzyme acyl-CoA ligase and transported into the mitochondria for /3 oxidation as we discuss later in this chapter. 

Substrate Enzyme Acylation agent Product Yield %) ee Value (%)... 

In another successful case, Hexter and Westheimer (1971) were able to locate 5% of the total radioactivity in Tyr-146 after irradiation of [14C]diazoacetylchymotrypsin. The reaction is actually intermolecular, occurring in chymotrypsin dimers. Westheimer s group have determined the structure of several of the modified amino acids derived from the photolysis of proteolytic enzymes acylated with diazo reagents. Such data is not available for other photoaffinity reagents. Knowing that O-carboxy-methyl tyrosine was an expected insertion product Hexter and Westheimer (1971) were able to show that of the two Tyr residues in the chymotrypsin B chain only Tyr-146, the C-terminal residue, was modified. If the nature of the modified amino acid had not been known it would have been considerably more difficult to pin-point the site of photolabeling. 

Khmelnitsky YL, Budde C, Arnold JM, Usyatinsky A, Clark DS, Dordick JS, Synthesis of water soluble paclitaxel derivatives by enzymic acylation, J. Am. Chem. Soc., 119 11554-11555, 1997. 

Cholesteryl esters that are internalized via the LDL receptor are hydrolyzed to produce cholesterol and an acyl chain. Cholesterol, in (urn, activates the enzyme acyl-CoA cholesterol acyl-transferase (ACAT) which re-esterifies cholesterol. In an apparently futile cycle, the cholesteryl esters are hydrolyzed by cholesteryl ester hydrolase. The cholesterol moiety has several fates it may leave the cell and bind to an acceptor such as high-density lipoprotein (HDL), it may be converted to steroid hormones, or it may be reesterified by ACAT. When the cellular cholesterol concentration falls, the activity of HMG-CoA reductase is increased, as is the number of LDL receptors, which results in an increase of cellular cholesterol, due both to de novo synthesis and to the uptake of cholesterol-rich lipoproteins in the circulation. An increase in cellular cholesterol results in the rapid decline in the mRNA levels for both HMG-CoA reductase and the LDL receptor. This coordinated regulation is brought about by the presence of an eight nucleotide sequence on the genes which code for both proteins this is termed the sterol regulatory element-1. 

A class of thiazole hydrazide inhibitors of cathepsin K has also been evaluated in references 26 and 27. Similar problems to those described in the preceding paragraph related to enzyme acylation and slow turnover of inhibitor in solution were observed in trying to determine the mechanism of protease inhibition based... 

Class I enzymes acylate the 2 -hydroxyl group of the terminal adenosine of tRNA, whereas class II enzymes (except the enzyme for Phe-tRNA) acyl-ate the 3 -hydroxyl group. 

Human leucocyte elastase (HE) is released in response to inflammatory stimuli and is responsible for degradation of connective tissues and is implicated in respiratory distress syndrome, rheumatoid arthritis, chronic bronchitis, and pulmonary emphysema. Many inhibitors of HE have been described [5], usually of the electrophilic carbonyl type, and often the key Val-Pro-Val motif has been exploited for potent inhibitory activity (3 - 5, Fig. 3). Recently, sivelestat (6, HE IC50 = 44 nM), a member of an enzyme-acylating series, has been approved in Japan for the treatment of acute lung injury [6]. 

Total Valence Energy for Steps in the Enzymic Acylation of ACh and ASCh (g-kcal/mole, relative to the reactants)... 

Retinaldehyde, when bound to retinol binding protein II (CRBPII), serves as a substrate for retinal reductase resulting in the production of retinol (14), which then binds to cellular retinol binding protein (CRBP) forming holo-CRBP. Holo-CRBP seems to be the preferred substrate for an esterification reaction (Fig. 7.6) mediated by lecithin retinol acyl transferase (LRAT), a microsomal enzyme that uses acyl groups donated from phosphatidylcholine (14).In cells not expressing CRBP, retinol esterification is carried out by a different enzyme, acyl CoArretinol acyl transferase (ARAT). 

The two steps are explained by the formation of a covalently bound enzyme- substrate intermediate (Figure 9,5). First, the acyl group of the substrate becomes covalently attached to the enzyme as p-nitrophenolate (or an amine if the substrate is an amide rather than an ester) is released. The enzyme-acyl group complex is called the acyl-enzyme intermediate. Second, the acyl-eni yme intermediate is hydrolyzed to release the carboxylic acid component of t ie substrate and regenerate the free enzyme. Thus, one molecule of p-nitrophenolate is produced rapidly from each enzyme molecule as the acyl-enzyme intermediate is formed. However, it takes longer for the enzyme to he reset by the hydrolysis of the acyl-enzyme intermediate, both steps are required for enzyme turnover. 

Huang et al. (1993) cite many studies that demonstrate that n-6 fatty acid are able to reduce the level of cholesterol in the blood serum. The data of Horrocks and Harder (1993) showed that n-6 fatty acids and n-3 fatty acids differ in their mode of action in cholesterol reduction, such that n-6 fatty acids redistribute cholesterol and n-3 fatty acid actually reduces the level of cholesterol. Davis (1992) demonstrated that n-3 essential fatty acids are more effective in reducing cholesterol levels in macrophages than n-6 essential fatty acids, most probably by the differential effect on the enzyme acyl-CoA (cholesterol acyltransferase). However, Horrocks and Harder (1983) indicated that cholesterol-esterifying enzymes that incorporate free fatty acids into cholesterol esters without the participation of CoA are also present in the rat brain. 

After calibration and measurement the currents were evaluated using a computer. In this manner, four measurement values from a complex sample could be obtained within 5 min without the performance of any separation step. The AMP sensor has been extended to the recognition of free fatty acids having a 6-10 carbon atom chain by coupling of acyl-CoA synthetase. In the reaction catalyzed by this enzyme acyl-CoA and AMP are liberated from fatty acids in the presence of ATP, CoA and Mg2+. AMP enters the four-enzyme sequence incorporated in the sensor. 

Non-enzymic Acylation, Deacylation and Migration. Traditionally, acetylation of a reducing sugar with acetic anhydride and fused sodium acetate gives predominantly the fully acetylated equatorial (usually p) pyranose, because of the greater acidity and nucleophilicity of the equatorial OH, whereas acid-catalysed acetylation gives the axial acetate, thermodynamically favoured by the anomeric elfect. 

The equilibrium of the enzyme acylation reaction can be shifted towards the synthesis of the amide by precipitation of the acylated product formed (Fig. 6). The racemic ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate 3 is an insoluble liquid, whereas the (R)-phenylacetamide 10 is an insoluble solid. The racemic ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate 3 was added to dilute hydrochloric acid. The pH of the reaction medium was then adjusted to 6. Phenylacetic acid (2 equiv.) was added and the pH of the medium was readjusted to 6. Soluble PGA (50 units/100 mg of racemic amine) was added, and the reaction was stirred at room temperature. After completion of the reaction, the pH of the reaction mixture was adjusted to 4. Filtration of the reaction mixture gave (R)-amide 10 in quantitative yield. Chiral HPLC analysis of this isolated amide showed the absence of (S)-amide. The pH of the filtrate was raised to 8, and the filtrate was extracted with ethyl acetate to obtain (S)-amine 11 (yield 90%) (Fig. 6). The chiral HPLC analysis indicated an R S ratio of 2 98. 

Acyl-activating enzyme. Acyl-CoA synthetase. Fatty acid thiokinase (long-chain). Lignoceroyl-CoA synthase. 

---