Ribonuclease cyclic phosphate intermediate

Bovine pancreatic ribonuclease catalyzes the hydrolysis of RNA by a two-step process in which a cyclic phosphate intermediate is formed (equation 16.35). The cyclization step is usually much faster than the subsequent hydrolysis, so the intermediate may be readily isolated. DNA is not hydrolyzed, as it lacks the 2 -hydroxyl group that is essential for this reaction. There is a strong specificity for the base B on the 3 side of the substrate to be a pyrimidine—uracil or cytosine. 

Fourteen years previously, Dawson et al. (1971) had observed cyclic inositol monophosphate (cIP) to be the major water-soluble product in enzyme-catalysed phosphatidyl inositol breakdown. Dawson observed that cIP and inositol 1-monophosphate (IP) were released from the enzyme simultaneously and surmised that the enzyme (PLC) was primarily a transferase, hydrolysing PI and PIPj by a mechanism similar to that of adenylate cyclase. The observation of 1,2-cyclic phosphate products does suggest that reaction proceeds via a cyclic phosphate intermediate, but an analogy is better drawn with the postulated mechanism of ribonuclease. In such a mechanism, the function of the 2-hydroxyl of phosphatidyl inositol (the only axial ring substituent) is similar to that of the 2 -hydroxyl of RNA in the reaction catalysed by ribonuclease (Scheme 47). Thus enzyme-catalysed phosphatidyl inositol breakdown can be seen as a two-step process, with cyclization (or transphosphorylation) followed by a hydrolysis... 

Ribonuclease U2 is a novel enzyme found in the culture broth of Ustilago sphaerogena (7, 106). Ribonuclease U2 splits, practically specifically, the phosphodiester bonds of purine nucleotides in RNA with the intermediary formation of purine nucleoside 2, 3 -cyclic phosphates, indicating the specificity is complementary to that of pancreatic RNase A (106). Like RNase N, RNase U2 very slowly hydrolyzes the intermediate, nucleoside 2, 3 -cyclic phosphate, to 3 -nucleotides (80, 106). Thus, RNase U2 is a useful tool, not only for the analysis of nucleotide sequences of RNA (90, 92, 107, 108) but also for the synthesis of various oligonucleotides containing adenylyl or guanylyl residue (30) (T. Koike, T. Uchida, and F. Egami, unpublished). 

The hydrolysis of RNA catalyzed by RNase A occurs in two distinct steps, with a 2, 3 -phosphate cyclic diester intermediate (see fig. 8.12). The intermediate can be identified relatively easily, because its breakdown is much slower than its formation. Ribonucleases do not hydrolyze DNA, which lacks the 2 -hydroxyl group needed for the formation of the cyclic intermediate. 

Ribonuclease A hydrolyzes RNA adjacent to pyrimidine bases. The reaction proceeds through a 2, 3 -phosphate cyclic diester intermediate. Formation and breakdown of the cyclic diester appear to be promoted by concerted general-base and general-acid catalysis by two histidine residues, and by electrostatic interactions with two lysines. These reactions proceed through pentavalent phosphoryl intermediates. The geometry of these intermediates resembles the geometry of vanadate compounds that act as inhibitors of the enzyme. 

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 1970, Eckstein and co-workers reported the first stereochemical study of an enzyme-catalyzed hydrolysis of a phosphate ester, the hydrolysis of the endo isomer of uridine 2, 3 -cyclic phosphorothioate (enrfo-cyclic UMPS) (72) by ribonuclease A (RNase A) 13). The hydrolysis of RNA catalyzed either by base or by RNase A proceeds by a two-step mechanism in which the 2 -hydroxyl group of a nucleotide unit within an RNA molecule acts as a nucleophile on the 3 -phosphodiester bond to displace the 5 -hydroxyl group of the neighboring nucleoside to form a 2, 3 -cyciic phosphate intermediate. RNase A then catalyzes the hydrolysis of this cyclic phosphate, mimicked by Eckstein s endo-cyclic UMPS, to yield the ultimate 3 -mononucleotide product. 

Mechanism. Ribonuclease is one of the most thoroughly studied of all enzymes. Its role is to catalyse the hydrolysis of RNA through cleavage of the Internucleotide phosphate. The mechanism of hydrolysis is seen as a two-step process. In the first step, a five-membered cyclic intermediate is formed by attack of the 2 -hydroxyl of RNA on phosphorus, displacing the 5 -oxygen of the adjacent nucleoside. The second step results in liberation of an RNA fragment with a free 3 -phosphate, by hydrolysis of the eyclic phosphate intermediate. Hydrolysis is usually a slower step and the intermediate may be readily isolated. 

Ribonucleases are the hydrolytic enzymes responsible for processing ribosomal RNA. For example, RNase A cleaves phosphodiester bonds of RNA at the 3 end of the pyrimidine nucleotides. The reaction is believed to occur in two discrete chemical steps involving a cyclic 2, 3 -phosphate intermediate. The RNase A reaction exhibits a pH rate profile that is optimal near pH 6. RNase A, as well as other ribonucleases (e.g., RNase B, RNase D, RNase E, RNase T) has been the target of extensive biochemical and biophysical research. 

Another type of bond that is ubiquitous in nature is phosphodiester bond making up the backbone of DNA or RNA. Enzymes can use more than one amino acid side chain in their active side for simultaneous bifimctional or multifunctional catalysis. Often an add-base catalyst is formed as in the enzyme ribonuclease A. The natural enzyme consists of 124 amino acids and catalyzes the hydrolysis of RNA phosphodiester bonds between the phosphorous atom and the 5 -oxygen atom. The mechanism of ester cleavage proceeds via a 2, 3 cyclo-phosphate intermediate. The histidine 119 and 12 function as acid and base to catalyze the formation of the cyclic intermediate while the lysine stabilizes the pentacoordinated transition state. The hydrolysis of the cyclic intermediate is then again catalyzed by both histidine residues (Figure 26). ... 

It is evident that ribonuclease and alkali digestion of RNA have much in common. However they differ in that ribonuclease possesses a pyrimidine specificity and hydrolyzes the intermediate cyclic phosphate to form only the 3 -phosphate. Alkaline hydrolysis in both of these steps is less specific. 

A portion of an RNA chain, indicating points of cleavage by pancreatic ribonuclease. Pyr refers to a pyrimidine Base can be either a purine or a pyrimidine. The 2, 3, and 5 carbon atoms are labeled. The enzymatic reaction proceeds in two steps, with a cyclic 2, 3 -phosphate diester as an intermediate. 

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 specificity of ribonuclease has been studied with small synthetic substrates. When diesters of various nucleotides are subjected to RNAase, only those compounds that are derivatives of pyrimidine nucleoside 3 -phosphate are hydrolyzed purine nucleotides and pyrimidine 2 - or 5 -phosphates are resistant to this enzyme. With both model substrates and ribonucleic acid, the action of RNAase has been shown to include the intermediate formation of cyclic nucleotides (III). ... 

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