Ribonuclease productive intermediates

Ribonucleoside 2, 3 -cyclic monophosphates are isolatable intermediates, and ribonucleoside 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. 

These reactions are the easiest to tackle, since they require only one phosphoryl oxygen to be substituted in both the substrate and the product. The classic example of this experiment is the first step in the hydrolysis of RNA catalyzed by bovine pancreatic ribonuclease. As discussed in detail in Chapter 16, ribonucle-ase catalyzes the hydrolysis of RNA by a two-step reaction in which a cyclic intermediate is formed. The stereochemistry of the first step (cyclization) (equation 8.35),... 

One alternative approach for demonstrating the existence of a complex during the course of a modification reaction is stereochemical in nature. For example, if enantiomers of a modification reagent give either different rates of modification or different products, the importance of multiple sites of interaction between the reagent and the protein, and hence intermediate complex formation is indicated. Examples of studies of this type include the alkylation of hovine pancreatic ribonuclease and papain by a variety of haloacids (Heinrikson et al. 1965 Eisele and Wallenfels 1968). 

Advantages of the solid-phase method are amenability to automation, the almost 100% yield of product for each reaction, the ease of removal of excess reagents and waste products by washing and filtration of resin particles, the lack of need for purification of intermediates, and speed. Peptides or proteins that have been synthesized by the solid-phase method include ribonuclease, bradykinin, oxytocin, vasopressin, somatostatin, insulin, and the S-chain of hemoglobin. The sequence analyses for these substances were confirmed by demonstrating that the synthetic products, constructed on the basis of sequence data, had the same biological activities as those of the corresponding natural substances. 

Other than ribonuclease, the enzymes we will be considering here all proceed by mechanisms in which the substrate has undergone covalent interaction with an active site nucleophile to form an intermediate species. In this section, we will examine the role of proton transfer in the enzyme-promoted hydrolysis of this intermediate to generate the second product of the reaction and liberate free enzyme. The mechanisms used by these enzymes all involve activation of the attacking water molecule by an active site residue participating as a general-base. 

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. 

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... 

Hydrolysis of RNA by crystalline pancreatic ribonuclease likewise proceeds through intermediate 2, 3 -cyclization 164)y but in this case the action is specifically limited to phosphoryl linkages associated with the pyrimidine nucleotides the cyclic intermediates subsequently are degraded by the enzyme only to the 3 -nucleotide type. Thus, the end-products are polynucleotides which terminate in 3 -pyrimidine nucleotide groups, and pyrimidine mononucleotides of the 3 -variety 165). The structural identification of many of the polynucleotides demonstrated that no simple alternating sequence of purines and pyrimidines exist in the intact RNA. 

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