Productive intermediates cyclic phosphate intermediate

Using the principles outlined in this article, the crystal structures of the following complexes of RNase A have been determined the free enzyme, both with and without a sulfate ion in the active site, the enzyme-dinucleotide complex, the enzyme-cyclic phosphate intermediate complex, the enzyme-transition state complex, and the enzyme-product complex, all at or near atomic resolution. This structural informa-... 

Active site, stochastic boundary simulations (Brooks and Karplus 1983 Briinger et al. 1985 Brooks and Karplus 1989) of RNase complexed with a substrate, a cyclic phosphate intermediate and a product have been made to clarify the contribution of various amino acid residues to the enzymatic reaction (Haydock et al. 1990). The structure of the enzyme complexed with a true substrate, CpA, was built from the crystal results for the enzyme complexed with deoxy-CpA, the substrate analog, by introducing the 02 H hydroxyl group to include the deprotonated His 12, the protonated His potential was replaced by that for the neutral form His 119 was protonated in the simulations. 

Certain RNases, such as RNase A, Tl, and U2, which cleave RNA via a 2, 3 -cyclic phosphate intermediate, require the presence of the 2 -OH group of ribose. In contrast to this class of RNases, RNase H has no mechanistic requirement for the 2 -OH group because the hydrolysis product has a 5 -P. Therefore the catalytic mechanism of RNase H, which is optimally active at pH 8, is rather unique and resembles those of some DNases. The catalytic groups of E. coli RNase HI have been identified as two Asp and one Glu residues that together form a unique carboxyl triad. ... 

The alcoholysis of the cyclic phosphate of catechol by alditols can lead, after acid hydrolysis of intermediate, cyclic phosphates, to the selective formation of phosphoric esters of the primary hydroxyl groups in the alditols. Thus, erythritol and D-mannitol afford, after chromatographic purification of the reaction products, their 1-phosphates in yields of 31 and 38%, respectively.217 The method was used to convert riboflavine into riboflavine 5 -phosphate.218 1-Deoxy-1-fluoro-L-glycerol has been converted into the 3-(dibenzyl phosphate) in 54% yield by selective reaction with dibenzyl phosphorochloridate. 219... 

RNase A utilizes a 2, 3 -cyclic phosphate ester as an intermediate (fig. 8.18) but yields only a 3 -phos-phate product. Within the mechanism, what controls the final position of the phosphate (i.e., why isn t the 2 -phosphate a product) ... 

Figure 10.17 Alkaline hydrolysis of RNA, showing only a single diester bond. The intermediate cyclic triester can be hydrolyzed at any two of the three locations indicated by dotted lines and labeled a, b, and c. If cleavage occurs at positions a and b, the first set of products is obtained. Cleavage at a and c produces the second set. A third set may be obtained by cleaving at b and c. P designates esterified phosphate.

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. 

A further intriguing aspect of the biological chemistry of cyclic inositol phosphates lies in the fact that they are not only detected as products of hydrolysis of PIPj catalysed by phospholipase C, but they may also be intermediates in this enzyme-catalysed reaction. The water-soluble products of the breakdown of PIPj catalysed by phospholipase C were hydrolysed in 0-labelled water in the presence of acid (Wilson et al., 1985a,b). The resulting trisphosphates after work-up were analysed by mass spectrometry-gas chromatography. Since only reactive cyclic phosphates are hydrolysed (with label incorporation) in dilute acid, Majerus and coworkers were able to use this technique to quantify the initial cyclic phosphate product of the phospholipase C reaction. Similar experiments were performed with phosphatidyl inositol 4-monophosphate (PIP) and phosphatidyl inositol (PI) as substrates and the results confirmed by hplc ananlysis. 

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... 

The chemical synthesis of cyclic nucleotides may be undertaken in a fashion analogous to their biosynthesis cyclization of an adenosine anhydride adduct. As the cyclic phosphate product is high energy, use of an anhydride intermediate, as may be obtained by reaction of a carbodiimide, will insure that cyclization is a thermodynamically allowable process. Hence, under conditions of high dilution and in the presence of DCC, adenosine... 

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