Adenosine cyclic 2 ,3 -phosphate hydrolysis

The nucleotide anhydride, adenosine 5 -triphosphate (24), when digested with aqueous barium hydroxide, gives a complex mixture containing such products as adenine, adenosine, adenosine 2 -, 3 -, and 5 -phosphates, adenosine 5 -pyrophosphate, and adenosine 2 (or 3 ),5 -diphosphate. - In addition, a nucleotide was foimd in this digest whose structure proved - to be that of adenosine 3 5 -cyclic phosphate (25). This component did not consume metaperiodate, and was degraded enzymically to adenosine 5 -phosphate (26) and adenosine 3 -phosphate (27), without the formation of adenosine 2 -phosphate. Hydrolysis of (25) with an acidic ion-exchange resin did, however, produce the 2 - and 3 -phosphates of adenosine. Compound (25) possessed only one phosphoryl dissociation, and showed a ratio of nucleoside to phosphate of 1 1, which, along with a molecular-... 

The nucleotide cyclic AMP (3, 5 -cyclic adenosine monophosphate, cAMP) is a cyclic phosphate ester of particular biochemical significance. It is formed from the triester ATP by the action of the enzyme adenylate cyclase, via nucleophilic attack of the ribose 3 -hydroxyl onto the nearest P=0 group, displacing diphosphate as leaving group. It is subsequently inactivated by hydrolysis to 5 -AMP through the action of a phosphodiesterase enzyme. 

Ribonuclease Ua digestion of ApU has revealed reduced hydrolase activity in the second step of RNase U2 action (30). When 87.4% of ApU was readily degraded to produce uridine and adenosine 2, 3 -cyclic phosphate, no 3 -adenylic acid was detected. After exhaustive degradation of ApU, hydrolysis of adenosine 2, 3 -cyclic phosphate occurred and 3 -adenylic acid gradually appeared. 

These were differently affected by different procedures. For example, when the enzyme was activated at 55°, the increment in ki was slight, but k2 increased 3.5-fold. Similarly, in the presence of EDTA, fc, and k2 values decreased independently, suggesting that the sites for both activities were different. Center and Behai (5) found that with the P. mirabilis enzyme, cyclic 2, 3 -UMP competitively inhibited the hydrolysis of bis(p-nitrophenyl) phosphate. The Ki was 40 pAf very close to the Km for the cyclic nucleotide (Km, 75 yM) which indicated that the two compounds could serve as alternate substrates being hydrolyzed at the same active site. In contrast, 3 -AMP was a mixed inhibitor of cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate hydrolysis. Adenosine was a mixed inhibitor of bis(p-nitrophenyl) phosphate hydrolysis but a competitive inhibitor of 3 -AMP hydrolysis. From such kinetic studies Center and Behai (5) suggested that two separate and adjacent sites A and B are involved in the hydrolysis of the diester and phos-phomonoester substrates. Site A serves as a binding site for hydrolysis of ribonucleoside 2, 3 -cyclic phosphates and together with site B catalyzes the hydrolysis of the diester bond. During this reaction 3 -... 

UMP becomes bound to site B which catalyzes the hydrolysis of the phosphomonoester bond. Adenosine and 3 -AMP by binding at site B could interfere with the breakdown of cyclic 2, 3 -UMP. Similarly, binding of bis (p-nitrophenyl) phosphate at site A could interfere with the breakdown of 3 -AMP. Cyclic 2, 3 -UMP and bis(p-nitrophenyl) phosphate compete for site A while adenosine competes with 3 -AMP for site B. Unemoto et al. (7) have examined the mutual inhibition of substrates and substrate analogs for the enzyme from halophilic V. alginolyticus. They also concluded that 3 -ribonucleotides and ribonucleo-side 2, 3 -cyclic phosphates are hydrolyzed at different sites. However, because of the nature of the mutual inhibition between 3 -AMP and bis(p-nitrophenyl) phosphate, they suggested that part of the site for the latter substrate overlaps with the 3 -nucleotidase site. At this time the precise mechanism of action of the enzyme is not settled, but clearly there are two active sites, one a 3 -nucleotidase site and a cyclic phosphate diesterase site. Anraku (18) has described this protein as a double-headed enzyme. 

An additional set of data lends some plausible support to the suggestion that RO" (in the reverse of step 1) and OH- attack different faces of the cyclic phosphate. The presence of adenosine, which is a good acceptor in the synthesis of dinucleotides from C>p (S91), also stimulates hydrolysis of C > p as shown by Wieker and Witzel (527) instead of competing for the phosphorus as might be expected if the mechanisms were the same in detail. 

In any event, it is clear that further study of the hydrolysis of nucleoside 3 5 -cyclic phosphates is necessary. It also remains to be explained why the 3 5 -cyclic phosphates of adenosine and guanosine are more resistant to glycosyl cleavage in acid than are the corresponding 5 -phosphates or nucleosides. 

Adenosine 3, 5 monophosphate (cyclic AMP) (R = adenine in (10.79b)) is of considerable importance in biochemistry. Hydrolysis of this compound with Ba(OH)2 gives a mixture of adenosine 3 phosphate and adenosine 5 phosphate. 

A similar kinetic analysis of the hydrolysis of cyclic adenosine 3, 5 -monophosphate (cAMP) by Ce(NH4)2(N03)6 indicated that the [Ce 2(OH)4]" + cluster is also the active catalytic species. This cyclic phosphate is an important biomaterial which is responsible for cell-to-cell communication. The hydrolysis of cAMP has many common features with DNA hydrolysis. First, both cAMP and DNA are enormously stable (the half-lives of the phospho-diester linkages in the absence of catalysts are estimated to be 400 thousand (Chin and Zou, 1987) and 200 million years, respectively). Second, the hydrolyses of both compounds are remarkably accelerated by Ce(IV) ion by lO -lO fold(Sumaokaetal., 1992,1994). Thirdly, only Ce(IV) is effective for these reactions. Fourthly, both reactions proceed via the attack by OH (or its relevant species) as external nucleophile. The only notable difference between them is the fact that cAMP is hydrolyzed more promptly than DNA because of its ring strain. These results further confirm the proposal that the dinuclear Ce(IV) cluster [Ce 2(OH)4] + should be responsible for the catalytic DNA hydrolysis. 

As usual, standard syntheses of nucleotides are not included in this survey. Using adenosine 5 -[(i )- 0, 0, 0]phosphate, it has been shown that the conversion of adenosine 5 -phosphate to cyclic AMP and the enzymatic hydrolysis of the latter to the former both occur with retention of configuration at phosphorus. Cyclic AMP can be used to prepare 2 -0-substituted adenosine derivatives 2 -0-succinyl adenosine was synthesized by acylation followed by enzymatic dephosphorylation on equilibration the 3 -0-succinyl isomer was slightly favoured (54%). ... 

Two groups have reported the synthesis of compounds of type (139) as potential models for ring-opening reactions of cyclic AMP. These studies included cases where the dioxaphosphorinane ring was both apical-equatorial and diequatorial, and conformations were studied by n.m.r.i98,i99 xhe predominant formation of 3 -monophosphates in the base-catalysed hydrolysis of nucleoside 3, 5 -cyclic phosphates has been interpreted in terms of the lone pair orientation effect, which may decrease the energy of the transition state for P-O bond-breaking.200 Conformational studies have been carried out on the diastereomers of adenosine cyclic 3 ,5 -phosphorothioate, where chair conformations predominate, and for the / p-isomer of deoxyadenosine cyclic phosphoranilidate, where a chair-twist equilibrium... 

The hydrolysis of adenosine 2, 3 -cyclic phosphate is catalysed by alkane 1,2-diamines. This has been explained in terms of intramolecular cooperation between the neutral and protonated forms of the diamine. ... 

Adenosine 2 - and 3 -monophosphate monomethyl and monoisopropyl esters undergo isomerization and hydrolytic cleavage to the 2, 3 -cyclic phosphate and the alcohol at comparable rates under acidic conditions, whilst at neutral pH, a pH-independent phosphate migration prevails.197 However the hydrolysis of the 2-chlorophenyl ester of uridine-3 -phosphate to 2-chlorophenol and uridine monophosphates is rapid compared with the phosphate migration under acidic conditions. 198 These studies have been extended to the interconversion and hydrolysis of 2 ,5 - and 3 ,5 -dinucleoside phosphates, where for adenosine systems, acid-catalysed depurination competed with phosphate migration and hydrolysis at pH<3.i99... 

Cells also contain nucleotides with phosphate groups in positions other than on the 5 carbon (Fig. 8 6). Ribonucleoside 2, 3 -cyclic monophosphates are isolatable intermediates, and rihonucleoside 3 -monophosphates are end products of the hydrolysis of RNA by certain ribonucleases. Other variations are adenosine 3, 5 -cyclic monophosphate (cAMP) and guanosine 3, 5 cyclic monophosphate (cGMP), considered at the end of this chapter. 

The 3, 5 -cyclic monophosphate of adenosine (cAMP) (2.148) is an important secondary messenger for intercellular communication in biochemistry. When the cell is stimulated by the first messenger, compound 2.148 is formed from adenosine triphosphate (ATP) (Scheme 2.25). This reaction is catalysed by an adenosine cyclase enzyme. The cAMP then goes on to activate other intracellular enzymes, so producing a cell response. The response is terminated by the hydrolysis of cAMP by phosphodiesterase (a phosphate-ester-hydrolysis enzyme). The action of adenylate cyclase has been mimicked successfully with a p-cyclodextrin complex of Pr(iii) and other lanthanide(iii) metals, under physiological conditions. The... 

SM2/AM1 and SM3/PM3 models were used to study the hydrolysis ofpyro-phosphate, which is coupled to virtually all biosynthetic reactions. However, the authors concluded that extreme care must be taken when applying semiempirical methods to compounds containing second-row atoms, since they may produce anomalously high atomic charges [98]. On die other hand, a study on syn and anti conformations of solvated cyclic 3 ,5 -adenosine monophosphate indicated that SM3/PM3 and SM2/AM1 models are inejqjensive yet accurate approaches... 

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