5 -Nucleotidase, action

CarbocycHc 2/3 -didehydro-2/3 -dideoxyguanosine [118353-05-2] (carbovk, CBV, 66), C H 2N502, synthesized in 1988 (177), is a promising candidate for the chemotherapy of AIDS. CBV inhibits HIV repHcation and HIV-induced cytopathic effects in a variety of human T-lymphoblastoid ceU lines at concentrations approximately two hundred- to four hundredfold below its cytotoxic concentrations (177). CBV is as effective as AZT and DDC in reducing the expression of vkal antigen in HIV-infected CEM ceUs (177). The antivkal potency and selectivity of carbovk is comparable to the anti-HIV-1 potency and selectivity of 2/3 -dideoxyadenosine (178). The exact mode of antivkal action of carbovk has not yet been elucidated, but may be the modulating effect of intraceUular nucleotides on 5 -nucleotidase activity (179). 

Purinergic System. Figure 2 Schematic of sympathetic cotransmission. ATP and NA released from small granular vesicles (SGV) act on P2X and a-i receptors on smooth muscle, respectively. ATP acting on inotropic P2X receptors evokes excitatory junction potentials (EJPs), increase in intracellular calcium ([Ca2+]j) and fast contraction while occupation of metabotropic ar-adrenoceptors leads to production of inositol triphosphate (IP3), increase in [Ca2+]j and slow contraction. Neuropeptide Y (NPY) stored in large granular vesicles (LGV) acts after release both as a prejunctional inhibitory modulator of release of ATP and NA and as a postjunctional modulatory potentiator of the actions of ATP and NA. Soluble nucleotidases are released from nerve varicosities, and are also present as ectonucleotidases. (Reproduced from Burnstock G (2007) Neurotransmission, neuromodulation cotransmission. In Squire LR (ed) New encyclopaedia of neuroscience. Elsevier, The Netherlands (In Press), with permission from Elsevier). 

Ribonucleoside 5 -0-hydroxymethylphosphonates (8 R = OH) are resistant to the action of phosphatases and phosphodiesterases. They are, however, good substrates for snake venom 5 -nucleotidase, unlike (8 R = H).2 ... 

Purine nucleotides are degraded by a pathway in which they lose their phosphate through the action of 5 -nucleotidase (Fig. 22-45). Adenylate yields adenosine, which is deaminated to inosine by adenosine deaminase, and inosine is hydrolyzed to hypoxanthine (its purine base) and D-ribose. Hypoxanthine is oxidized successively to xanthine and then uric acid by xanthine oxidase, a flavoenzyme with an atom of molybdenum and four iron-sulfur centers in its prosthetic group. Molecular oxygen is the electron acceptor in this complex reaction. 

AMP can be converted by the action of AMP 5-nucleotidase to adenosine (step c, Fig. 25-17), which is thought to be an important local hormone or second... 

A specific protein inhibitor for 5 -nucleotidase has been purified from E. coli cell cytoplasm (10, 16). It prevents the action of the enzyme on 5 -AMP, ATP, and UDPG. It also inhibits the hydrolysis of 5 -AMP by the 5 -nucleotidases from A. aerogenes, S. sonnei, and S. typhimurium (10). Other Enterobacteriaceae also possess similar intracellular protein inhibitors (18) which inhibit all hydrolytic activities of the 5 -nucleo-tidase of these organisms. The relevance of this inhibitor protein to the action of the enzyme in vivo is not known. 

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. 

Fig. 4 Mechanisms involved in the extracellular inactivation of nucleotides (a, b and c) and adenosine (d) and their influence on purine concentration in the P2Y and PI receptor biophases, (a) NT-PDasel hydrolyses ATP and ADP very efficiently, thus preventing their action on P2Y receptors (b) NTPDase2 metabolizes ATP preferentially, allowing an accumulation of ADP and thus favouring activation of P2Yi, 12,13 receptors (c) NTPDase3 hydrolyses both ATP and ADP slowly, giving them time to activate both P2Y2,4 and P2Y 1,12,13 receptors. Formation of adenosine depends on the activity of ecto 5 -nucleotidase (CD73). Adenosine inactivation systems also influence adenosine concentration in the PI receptor biophase (d) the nucleoside transporters take up adenosine adenosine deaminase (ADA) regulates both the concentration of adenosine in the Ai receptor biophase and the functionality of Ai receptors.

Purines and pyrimidines in excess of cellular requirements can be degraded. The extent of degradation depends on the organism. Humans cannot degrade purines beyond uric acid because we lack the enzyme uricase, which splits the purine ring to form allantoin. In humans excess AMP is deaminated to IMP by the action of a specific deaminase. IMP is then hydrolyzed by 5 -nucleotidase to form inosine. Inosine and guanine are oxidized to urate as follows ... 

Gulland and Jackson performed some experiments with 5-nucleotidase, a highly specific enzyme which dephosphorylates 5-phospho-adenosine and -inosine but not" 5-phospho-guanosine and -uridine it is apparently not yet known whether the enzyme dephosphorylates 5-phos-pho-cytidine. They found that a mixture of phosphodiesterase with 5-nucleotidase liberates 35% of the total phosphorus as inorganic phosphate, and therefore decided that two or more of the phosphoryl groups may be attached at position (5) of the ribose units. The 35% dephosphorylation, intermediate between 25 and 50%, was explained as the result of simultaneous, competitive diesterase action at A and B, on two or more phosphoryl groups ... 

In 1933, Klein discovered that sodium arsenate inhibits the action of the nucleotidase" of intestinal mucosa. Consequently, Klein and Thannhauser were enabled to hydrolyze thymus nucleic acid by means of intestinal desoxyribonucleinase without subsequent dephosphorylation of the liberated nucleotides. Making use, also, of Klein s discovery of the deaminase-inhibiting activity of silver ions, they were successful in isolating the adenine nucleotide. Hence the phosphodesoxyribosyl nucleotides of adenine, guanine, thymine, and cytosine were isolated and characterized. 

Catabolism of the nucleotides (Figure 24-3, B) begins with removal of their ribose-linked phosphate, a process catalyzed by purine 5 -nucleotidase. Removal of the ribose moiety of inosine and guanosine by the action of purine-nucleoside phosphorylase forms hypoxanthine and guanine, both of which are converted to xanthme. Xanthine is converted to uric acid through the action of xanthine oxidase. 

Methylglutaryl-CoA hydratase (EC 4.2.1.18) catalyzes the reversible dehydration of HMG-CoA into 3-methylglutaconyl-CoA and is known from mammalian cells to be involved in leucine metabolism. This enzyme was detected in C. roseus suspension cultured cells and partially purified (87). Further enzymes channeling HMG-CoA away from isoprenoid biosynthesis in C. roseus cells were detected by a selective HPLC system showing that the CoA-esters may be rapidly dephosphorylated on the 3 -position by the action of 3 -nucleotidases (87). 

Two publications on the higher ketose mono- and bis-phosphates in rat-liver extracts have described their detection and estimation by colorimetry and enzymic assay,and the synthesis of octulose 1,8-bisphosphates using muscle aldolase. A stereospecific synthesis of adenosine 3, 5 -cyclic phosphothioate has appeared/ Standard condensation methods have been used to synthesize the 5 -O-(Taminoethane-phosphonyl) nucleosides (21) and (22). The compounds were shown to be inert to the action of alkaline phosphatase and are poor substrates for 5 -nucleotidase. The selenophosphates (23) and (24) have been prepared.Phosphorylation of nucleosides using dibenzyl hydrogen phosphate,... 

Two enzymes were selected In which proper alignment of nucleotide C3 and base moieties Is necessary for their catalytic action AMP amlnohydrolase (AMP deaminase, EC 3>3.4.6) from rabbit muscle requires substrates with an anion at C5 and catalyses deamination at C6-NH2 of adenine [42]. Vice versa, In snake venom 3 -nucleotidase (EC 3>1 3.5 hydrolysis occurs at the 5 -phosphate vdilch has to be at a defined distance from the base, as Judged by the Inactivity of 3 -phosphates [43]. 

Although several enzymes can in theory convert purine ribonucleotides to ribonucleosides, in only a few cases is it known exactly which is acting. Certain strains of Bacillus svbtilis accumulate nucleotides extra-cellularly, but mutants lacking 5 -nucleotidase or alkaline phosphatase, or both, accumulate smaller quantities of nucleotides than cells which have both enzymes. 5 -Nucleotidase appeared to be quantitatively the more important for nucleotide dephosphorylation in these cells 23). Baer and Drummond 12) compared the rates of dephosphorylation of the 2 -, 3 -, and 5 -phosphates of adenosine by perfused rat heart and concluded that only a specific 5 -nucleotidase was actively in contact with the blood. More evidence for the action of specific, rather than nonspecific phosphatases, are the observations that in one system or another, adenylate, inosinate, xanthylate, or guanylate appears to be the nucleotide most rapidly de-phosphorylated. 

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