Guanosine 3 :5 -cyclic phosphate

Most of the properties of RNase Ti are summarized in Tables II and IV. It is a very acidic protein, active between pH 4 and 8.5 it is most active at pH 7.5 for RNA digestion (12) and at pH 7.2 for the hydrolysis of guanosine 2, 3 -cyclic phosphate (18). The purified enzyme possesses a specific activity of about 1.6 X 10 units/mg of protein. The molecular activity (standard units/jumole enzyme) has not been determined for the cleavage of a definite dinucleoside monophosphate such as GpC or for the hydrolysis of guanosine 2, 3 -cyclic phosphate. 

Unlike guanosine 2, 3 -cyclic phosphate, guanosine 3, 5 -cyclic phosphate is quite resistant to the enzyme (S. Sato, T. Uchida, and F. Egami, unpublished). 

The sugar specificity of RNase Tx appears to require a 2 -hydroxyl group for the substrate because DNA is not attacked by RNase Tx. This is consistent with the intermediary formation of 2, 3 -cyclic phosphate and also with the finding that 2 -0-methylated guanylyl bonds in tRNA is resistant to the enzyme (48)- Holy and Sorm (49) found that RNase Tx did not attack L-guanosine 2, 3 -cyclic phosphate and L-inosine 2, 3 -cyclic phosphate. They found further that RNase Tx split 9-(a-L-lyxo-furanosyl)-hypoxanthine 2, 3 -cyclic phosphate but not the D-lyxofura-nose derivative, and they concluded that the substrate molecule was fixed at least to three regions of RNase Tx (50). 

Various experimental evidence suggests that only 2 or 3 of the 9 tyrosine residues are on the surface of the enzyme (19, 55). Indeed only a part of the tyrosine residues can be easily modified by acetylimidazole at pH 7.5 or by tetranitromethane at pH 8.0 (H. Kasai, K. Takahashi, and T. Ando, unpublished). As enzymes thus modified have catalytic activity, the tyrosine residues that are probably located at the surface of the enzyme do not seem to be essential for activity. Consistent results were also obtained from the modification by fluorodinitrobenzene or by diazo-lH-tetrazole (H. Kasai, K. Takahashi, and T. Ando, unpublished). Especially noteworthy is the derivative, in which one to two tyrosine residues, amino terminal alanine, and one lysine residue were modified with diazo-lH-tetrazole. The derivative was deprived of most of its activity toward RNA but retained about 50% of its activity toward guanosine 2, 3 -cyclic phosphate. This may be explained by some steric hindrance owing to the modification of a tyrosine residue near the active center. 

RNA and the activity decreases steeply in alkaline medium. The pH dependence of hydrolase activity for guanosine 2, 3 -cyclic phosphate is not yet determined. The crystals of RNase Nj possess a specific activity of about 4.0 X 104 units/mg of protein, about 2.5 times that of purified RNase Ti. 

When guanosine 2, 3 -cyclic phosphate is incubated with about 3-fold nucleoside at a low temperature in the presence of RNase N, guanylyl-(3, 5 )-nucleoside can be obtained as a synthetic product. For example, using uridine as a phosphate acceptor, GpU was obtained with a high yield of 27% calculated upon guanosine 2, 3 -cyclic phosphate added. This... 

Several other phosphodiesterases have been described, such as ri-bonuclease Ti from takadiastase. This enzyme hydrolyzes RNA and the RNA core produced by pancreatic ribonuclease, with the rapid liberation of guanosine 3 -phosphate and guanosine 2, 3 -cyclic phosphate. A second enzyme from the same source, ribonuclease Tj, rapidly liberates adenylic acid and pyrimidine mononucleotides while guanosine 3 -phosphate is liberated slowly (161). 

RNase Tl cleaves P-05 ester bonds in ssRNA, specifically at the 3 -P of the guanylic acid residues. As in RNase A (see Fig. 3.3), the catalysis occurs by a two-step mechanism, i.e., the formation of a terminal guanosine 2, 3 -cyclic phosphate intermediate (transesterification step) and the hydrolysis of the cyclic ester to guanosine 3 -monophosphate (hydrolysis step). The transesterification step involves a general acid—base catalysis. 

The optimum pH values of RNase Ta for the hydrolysis of guanosine cyclic phosphate and xanthosine cyclic phosphate are pH 7.2 and 4.5, respectively. The xanthosine cyclic phosphate may have the lactam form (-NH-CO-) susceptible to the enzyme only in the acidic medium (18). 

Figure 12-4. IR-UV double resonance spectrum of guanosine-cyclic-phosphate...

Tegeder, I., Schmidtko, A., Niederberger, E., Ruth, P., and Geisslinger, G. (2002). Dual effects of spinally delivered 8-bromo-cyclic guanosine mono-phosphate (8-bromo-cGMP) in formalin-induced nociception in rats. Neurosci. Lett. 332, 146—150. 

The purine ring system is undoubtedly among the most ubiquitous of all the heterocyclic compounds. This arises not only from the universal occurrence of adenine and guanine in DNA and RNA and of additional modified derivatives in the various tRNAs but also from the subsidiary uses of the ring system in very many biochemical systems Indeed across the whole spectrum of biochemical reactions in living systems there is hardly a reaction sequence which does not involve in some way a purine derivative such as the adenosine or guanosine mono-, di- and tri-phosphates, associated cyclic phosphates and nucleotide coenzymes. 

Cyclic phosphates of adenosine and guanosine are regioselectively cleaved to 2 -phosphates at pH 11.0 using 3 and y-cyclodextrins as catalysts. 

Adenylic acids o and b were each converted by trifluoroacetic anhydride into the same 2 3 -cyclic phosphate (13, R = adenin-9-yl) by Brown, Magrath, and Todd. These authors also applied this method to the synthesis of 2 3 -cyclic phosphates (13) of cytidine, uridine, and guanosine. Hydrolysis of these cyclic phosphates gave the corresponding a and b nucleotide mixtures. Markham and Smith found that, during hydrolysis of ribonucleic acid with pancreatic ribonuclease, the 2 3 -cyclic phosphates of the pyrimidine nucleosides are formed as intermediates leading to the ribonucleoside phosphates b. The also showed that 2 3 -cyclic phosphates (13) are formed by very mild, alkaline hydrolysis of ribonucleic acid. The discoveries of these a and b isomers of mononucleotides from... 

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. 

Abbreviations used NAD+ = nicotinamide adenine dinucleotide NADH e reduced nicotinamide adenine dinucleotide NADP = nicotinamide adenine dinudeotide phosphate NAD PH reduced nicotinamide adenine dinucleotide phosphate NMN, NMN+ nicotinamide mononucleotide NMNH2 = reduced nicotinamide mononucleotide a-NAD a-nicotinamide adenine dinucleotide AMP = 5 -adenylic acid 3,5 -AMP adenosine 3, 5 -cycIic phosphate 3 ,5 -UMP = uridine 3, 5 -cyclic phosphate 3, 5 -CMP cytidine 3, 5-cyclic phosphate 3 f5 GMP = guanosine 3 5f-cyclic phosphate 3, 5 TMP thymidine 3, 5 -cyclic phosphate Dibutyryl-3, 5 -AMP = N6,02-dibutyryladenosine 3, 5 -cyclic phosphate 2, 3 -UMP = uridine 2 ,3 -cyclic monophosphate 2, 3 -CMP cytidine 2, 3 -cyclic monophosphate 2, 3 -AMP = adenosine 2, 3 -cyclic monophosphate 2 ,3 -GMP = guanosine 2 3 -cyclic monophosphate 2 -UMP = uridine 2 -phosphate -UMP uridine -phosphate 5 -UMP = uridine 5 phosphate Poly U polyuridylic acid ADP = adenosine 5 -diphosphate FAD = flavin adenine dinucleotide UpA, UpU, ApU and ApA x dinucleoside phosphates of uridine and/or adenine. c See original references for experimental conditions and additional data. 

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