Dinucleotide, hydrolysis

The turnover reaction of hydrolysis of 2, 3 -CMP could be made negligibly slow at temperatures below -60°C at pH 3-6 in 70% methanol, and below -35°C at pH 2.1. The rate of the catalytic reaction using crystalline enzyme was found to be 50-fold slower than that of dissolved enzyme for cyclic phosphate hydrolysis, and 200-fold slower for dinucleotide hydrolysis (presumably the greater reduction for the larger substrate reflects increased diffusional hindrance by the small solvent chan-... 

While formation of a dinucleotide may be represented as the elimination of water between two monomers, the reaction in fact strongly favors phosphodiester hydrolysis. Phosphodiesterases rapidly catalyze the hydrolysis of phosphodiester bonds whose spontaneous hydrolysis is an extremely slow process. Consequently, DNA persists for considerable periods and has been detected even in fossils. RNAs are far less stable than DNA since the 2khydroxyl group of RNA... 

The conformations of L-adenylyl-(3 5 )-L-adenosine (28) and L-adenylyl-(2 -> 5 )-L-adenosine (29), as deduced from circular dichroic spectra, are different from the corresponding DD-dinucleotides. < The n.m.r. and u.v. absorption spectra of (28) and (29) are the same as the DD-dimers and their chromatographic and electrophoretic properties appear identical. While (28) and (29) are resistant to enzymic hydrolysis they form complexes with polyU. 

Second, the efficiency of exonucleases and endonucleases in hydrolysing DNA is greatly affected by modification of the bases (Dizdaroglu et /., 1978 Breimer, 1991). For example, Maccubbin et al. (1991) reported that the presence of 8-OH-Gua severely inhibits digestion of dinucleotides by phosphodiesterase. Thus it is not always certain that modified bases are completely hydrolysed from DNA, especially when published hydrolysis techniques are transplanted from one laboratory to another and not revalidated. 

Gill, D.M., Pappenheimer Jr, A.M, Brown, R., and Kurnick, J.T. (1969) Studies on the mode of action of diphtheria toxin VII. Toxin-stimulated hydrolysis of nicotinimide adenine dinucleotide in mammalian cell extracts./. Exp. Med. 129, 1-21. 

Disulfoton causes neurological effects in humans and animals. The mechanism of action on the nervous system depends on the metabolism of disulfoton to active metabolites. The liver is the major site of metabolic oxidation of disulfoton to disulfoton sulfoxide, disulfoton sulfone, demeton S-sulfoxide and demeton S-sulfone, which inhibit acetylcholinesterase in nervous tissue. These four active metabolites are more potent inhibitors of acetylcholinesterase than disulfoton. Cytochrome P-450 monooxygenase and flavin adenine dinucleotide monooxygenase are involved in this metabolic activation. The active metabolites ultimately undergo nonenzymatic and/or enzymatic hydrolysis to more polar metabolites that are not toxic and are excreted in the urine. 

Figure 6.4. Schematic representation of a fluorescent immunoassay for theophylline utilizing enzymatic hydrolysis of an intramolecularly quenched theophylline conjugate of flavin adenine dinucleotide. (Reprinted from Ref. 5, with permission from Academic Press.)...

Riboflavin is the redox component of flavin adenine dinucleotide FAD. It is derived from FAD by hydrolysis of a phosphate ester link. The fully oxidised form of FAD is involved in many dehydrogenaze reactions during which it is converted to the fully reduced form. The fully oxidised state is restored either by another redox enzyme or by interaction with oxygen and hydrogen peroxide is liberated. The one-electron reduced, semiquinone form of FAD, is involved in some electron transfer steps. 

ESI-MS has been used for the quantification of a number of substrates and products of enzymatic reactions [56,57]. Hsieh et al. report the use of ion spray mass spectrometry (a technical variation of electrospray ionization) coupled to HPLC for the kinetic analysis of enzymatic reactions in real time [58]. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A and the hydrolysis of lactose with 3-galactosidase were monitored and the resulting data were used for the estimation of and v x of these reactions. Another field of application of electrospray mass spectrometry is the screening of combinatorial libraries for potent inhibitors [31,59]. 

Weinblum and Johns43 also have shown that hydrolysis of the two dimeric dinucleotide products, D1 and Dz, leads to the isolation of two forms of thymine dimer which were assigned tentatively to structures 10 and 11. 

Draw the structure of a dinucleotide that might be obtained by the partial hydrolysis of RNA. Indicate the following ... 

The hydrolysis of dinucleotides by the staphylococcal nuclease has been recently studied in Laskowski s laboratory (12). In 12 dinucleotides of the type d-N pN p the susceptibility to hydrolysis varied about 100-fold and appeared to depend primarily on the type of base in the p position. In addition, the corresponding d-NpN dinucleosidemonophos-phates were more resistant to hydrolysis, and the pNpN 5 -phosphodinu-cleotides were most resistant of all. This work would appear to be consistent with our view of the pdTp binding site of the nuclease at high resolution. 

Although the contribution of the purine and pyrimidine bases is considered to be relatively minor compared to the electrostatic effects of the phosphates, the enzymic subsites must clearly be able to preferentially distinguish and discriminate the various bases. For example, Mikulski et al. have shown that the nature of the base in the / position exerts a dominant role in the susceptibility of hydrolysis of dinucleotides a clear preference is demonstrated for A and T (22). Also consistent with this are the observations that 5 -mononucleotide binding shows a clear preference for A and T (3, 63), that poly A is more rapidly hydrolyzed than poly C or poly U (3), and that Tp are preferentially released during the early phases of RNA and DNA hydrolysis. 

In Fig. 2 the ratio of the rates of hydrolysis of phosphorylated to that of dephosphorylated dinucleotides is plotted vs. pH. The ordinate is on the logarithmic scale. The ratio changes from about 40 at pH 5 to about 1700 at pH 9. [The previous estimate of the latter value (as 1000/10 = 100 in the tabulation) was an underestimate.]... 

The X-ray data do not yet, and cannot conclusively, provide an answer to this question. The current interpretation does distinctly allow for the possibility that transphosphorylation occurs by an opposite attack (see Section VI,G,3) and that hydrolysis occurs by an adjacent attack. The dinucleotide phosphonate as depicted in Fig. 23 seems poised for an attack of 02 opposite C5"-CH2 while W, is in good position to make an... 

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. 

As mentioned briefly above, the enzymatic excision of damaged nucleobases may cause some problems. A case in point is the action of nuclease PI. While a single 8-oxo-G lesion is excised as the damaged nucleoside, the clustered 8-oxo-G/Fo lesion is only obtained as modified dinucleotide (Maccubbin et al. 1992). Another example is the hydrolysis of dG pC which severely inhibits the action of bovine spleen phosphodiesterase, while HMUrapA shows only very little inhibition (Maccubbin et al. 1991). Enzymatic hydrolysis of DNA is, in fact, the recommended method for the determination of HMUra (Teebor et al. 1984 Frenkel et al. 1985). It is recalled that mammalian cells cope with this DNA lesion with the help of a hydroxymethyluracil glycosylase (Hollstein et al. 1984). 

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