Acyl phosphates, synthesis

Based on a series of studies of the effect of organic solvent on the reaction of Ca-ATPase with Pj and ATP synthesis, De Meis et al. proposed that a different solvent structure in the phosphate microenvironment in Ej and E2 forms the basis for existence of high- and low-energy forms of the aspartyl phosphate [93]. Acyl phosphates have relatively low free energy of hydrolysis when the activity of water is reduced, due to the change of solvation energy. The covalently bound phosphate may also reside in a hydrophobic environment in E2P of Na,K-ATPase since increased partition of Pj into the site is observed in presence of organic solvent [6] in the same manner as in Ca-ATPase. 

In both cases, the mixed anhydride is used to synthesize ATP from ADP. Hydrolysis of the anhydride liberates more energy than the hydrolysis of ATP to ADP and, therefore, can be linked to the enzymic synthesis of ATP from ADP. This may be shown mechanistically as a hydroxyl group on ADP acting as nucleophile towards the mixed anhydride, and in each case a new phosphoric anhydride is formed. In the case of succinyl phosphate, it turns out that GDP rather than ADP attacks the acyl phosphate, and ATP production is a later step (see Section 15.3). These are enzymic reactions therefore, the reaction and the nature of the product are closely controlled. We need not concern ourselves why attack should be on the P=0 rather than on the C=0. 

We have already described the biosynthesis of glutamate and glutamine. Proline is a cyclized derivative of glutamate (Fig. 22-10). In the first step of praline synthesis, ATP reacts with the y-carboxyl group of glutamate to form an acyl phosphate, which is reduced by NADPH or NADH to glutamate y-semialdehyde. This intermediate undergoes rapid spontaneous cyclization and is then reduced further to yield proline. 

Figure 17-7 Outline of the glycolysis pathway by which hexoses are broken down to pyruvate. The ten enzymes needed to convert D-glucose to pyruvate are numbered. The pathway from glycogen using glycogen phosphorylase is also included, as is the reduction of pyruvate to lactate (step 11). Steps 6a-7, which are involved in ATP synthesis via thioester and acyl phosphate intermediates, are emphasized. See also Figures 10-2 and 10-3, which contain some additional information.

A much more general method for acyl silane synthesis involving silyl diazo intermediates is illustrated in Scheme 1688. The lithiated derivative of trimethylsilyl diazomethane reacts smoothly with alkyl halides in THF solution to give a-trimethylsilyl diazoalkanes in good yields. Oxidative cleavage of the diazo moiety is effected using 3-chloroperbenzoic acid in benzene solution, to give access to a wide variety of acyl silanes in yields of up to 71%. A phosphate buffer (pH 7.6) is used to prevent side reactions. Aromatic acyl silanes clearly cannot be prepared by this chemistry since an aromatic nucleophilic substitution reaction would be required. 

Answer Activation of carboxyl groups by ATP could in theory be accomplished by three types of reactions the formation of acyl-phosphate + ADP of acyl-ADP + P, or of acyl-AMP + PP,. All these reactions are readily reversible. To create an activation reaction with a highly negative AG ° (effectively irreversible), the third type of reaction can be coupled to a pyrophosphatase reaction, as in the synthesis of fatty acyl-CoA molecules. 

Stadtman, E. R. and H. A. Barker Fatty acid synthesis by enzyme preparations of Clostridium kluyveri. VI. Reactions of acyl phosphates. J. Biol. Chem. 184, 769-793 (1950). 

Methanococcus voltae contains a membrane-bound vanadate-sensitive ATPase [48] that is inhibited by diethylstilbestrol, an inhibitor of eukaryotic P-type ATPases. The purified enzyme is composed of a single subunit (Mr 74 000), forms a covalent acyl-phosphate enzyme intermediate, and is not inhibited by nitrate or bafilomycin [49]. No such ATPase activity has been reported in other archaea. The presence of a second ATPase in M. voltae has been inferred since membranes react with antiserum prepared against the 3 subunit from the V-type ATPase of S. acidocaldarius [50]. Two peptides are detected whose Mr values (51 000 and 65 000) correspond to the masses for the two laigest subunits of the S. acidocaldarius ATPase [51]. There is evidence that ATP synthesis in the M. voltae enzyme is due to the operation of a sodium-translocating ATPase [50]. The relationship of the putative V-like ATPase to the sodium-translocating ATPase has not been established. 

Tang, K.-C., and Coward, J.K.. Synthesis of acyl phosphonate analogues of biologically important acyl phosphates. A-(2-Amino-10-methylpteroyl)-5-amino-2-oxopentanephosphonic acid, J. Org. Chem., 48, 5001, 1983. 

Thompson, J.E., Enol and acyl phosphates as intermediates in the synthesis of nonrandom triglycerides, J. Org. Chem., 30, 4276, 1965. 

The acyl-phosphate group at position 1 is much more energy rich than the phosphate anhydride of ATP, so 1,3BPG has the energy to drive synthesis of ATP from ADP + Pi, which it does in the glycolysis reaction catalyzed by the enzyme phosphoglycerate kinase. 

Acetylphosphate CH3 COOPO(OH)2, a high energy acyl phosphate. It is the product of acetate activation in some organisms Acetate <- ATP A. p. -I- ADP, a reaction catalysed by acetate kinase (EC 2.7.2.1). The back reaction is sometimes exploited for ATP synthesis, e.g. in the phosphoroclastic fission of pyruvate. 

Nucleoside analogues require phosphorylation to the 5 -triphosphate by cellular kinases prior to their inhibition of DNA synthesis. However, most studies have focused upon prodrugs of monophosphates, whereas for some nucleosides (for example, AZT) the formation of the diphosphate is rate limiting. Huynh-Dinh and coworkers [109, 110] have recently prepared P-acyl esters of 5 -di- (m = I) and tri-phosphates (m = 2) of both AZT and d4T (97, n = 6, 10, 12 or 14, Nu = 5 -AZT or 5 -d4T). The lipophilic acyl chain should facilitate passive diffusion across cellular membranes. Chemical hydrolyses at 37°C and physiological pH showed that the acyl phosphates were cleanly converted to the corresponding nucleoside di- and tri-phosphates. 

The spontaneous synthesis of the acyl phosphate, E-P, is analogous to the synthesis of ATP from ADP and Pi by the Fi ATPase and by myosin, except that in this case one component of the reaction becomes covalently bound to the enzyme. The energetics of the system at pH 7 in the presence of 5 mM Mg " and 0.1 M KCl is illustrated by Fig. 4 [31]. Experimentally, the binding of Pj to the enzyme is downhill by - 1.9 kcal mol" and the synthesis of E-P is slightly more downhill by - 0.3 kcal mol The synthesis of an acyl phosphate from inorganic phosphate and the carbox-ylate group of acetate (10) is uphill by 10.3 kcal mol" at pH 7. Formation of the acyl phosphate at the active site will be uphill by the same amount if the carboxyl... 

Since thiol estem are the immediate end products of various oxidative reactions, the phosphorolysis of these intermediates constitutes a mechanism by which the sulfhydryl enzymes and coenzymes involved are constantly regenerated with the simultaneous formation of more generally useful energy-rich acyl-phosphate compounds. In the presence of specific enzymes the phosphoryl groups of the acyl phosphates are transferred to ADP with the formation of ATP. For example, many bacteria contain an enzyme that catalyzes the synthesis of ATP by a reaction between acetyl-P and ADP [reaction (15)]. The enzyme catalyzing this reaction... 

Although the schemes presented in Fig. 4 represent one important mechanism of coupling substrate oxidation with phosphorylation in bacteria, other mechanisms may occur also. In higher animals, phosphotransacetylase and phosphotranssuc-cinylase appear to be absent. In these organisms, therefore, the acyl-SCoA derivatives formed in the oxidation of pyruvate and a-ketoglutarate are used to synthesize ATP by mechanisms that do not involve the intermediate formation of acyl phosphate. ATP formation from acetyl-SCoA (11) appears to occur by reactions (1), (2), and (3), whereas the synthesis of ATP from succinyl-SCoA occurs by an as yet undetermined pathway (15). 

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