Ribonuclease enzymic cleavage

The next section describes further mimics of ribonuclease enzymes. However, we mention here one additional study on the cleavage of ribonucleotides in which metal ions were used [123]. Cyclization of uridyluridine 39, and hydrolysis of the resulting cyclic nucleotides 40, was catalyzed by Eu3+ much more effectively than by Zn2+, and some added ligands increased the rates. 

Ribonuclease II [EC 3.1.13.1], also called exoribo-nuclease II, catalyzes the exonucleolytic cleavage of the polynucleic acid, preferring single-stranded RNA, in the 3 - to 5 -direction to yield 5 -phosphomononucleotides. The enzyme processes 3 -terminal extra-nucleotides of monomeric tRNA precursors, following the action of ribonuclease P. Similar enzymes include RNase Q, RNase BN, RNase PHI, and RNase Y. Ribonuclease T2 [EC 3.1.27.1] is also known as ribonuclease II. 

This enzyme [EC 3.1.26.6], also known as endoribo-nuclease IV and poly(A)-specific ribonuclease, catalyzes the endonucleolytic cleavage of poly(A) to fragments terminated by 3 -hydroxyl and 5 -phosphate groups. Oligonucleotides are formed with an average chain length of ten. 

Another technique, RNase cleavage (M4), uses the enzyme ribonuclease A to cut RNA-DNA hybrids wherever there is a mismatch between a nucleotide in the RNA... 

The ribonucleases are a class of enzymes catalyzing the hydrolytic cleavage of ribonucleic acids. Although such activity can be demonstrated in almost all tissues both plant and animal, relatively few of... 

The apparent usefulness of the modeling approach suggested that possible active site interactions important in understanding the mode of action of the well-characterized enzymes, ribonuclease (16) and staphylococcal nuclease (17). may be revealed. Both have been the subject of extensive crystallographic studies (18,19) with suitable inactive substrates in place. We considered the first step of hydrolytic action of ribonuclease (RNase) on the dinucleotide substrate uridylyl-(3 -5 )-adenosine(UpA). Our results (20) on the enzyme mechanism were consistent with the main features summarized by Roberts et al (21). The first step is a transphosphorylation leading to cleavage "oT the phosphodiester... 

With the existence of this new cyclodextrin lock, it was again important to select a key to fit it and to serve as substrate. For this we wanted a cyclic phosphate ester that this cyclodextrin bisimidazole could hydrolyze. The enzyme ribonuclease hydrolyzes cyclic phosphates as the second step in the hydrolysis of RNA, and cyclic phosphates are used as assay substrates for the enzyme. The advantage to us of such a substrate was that the geometry of the transition state would be relatively well-defined, so that it should be possible to design congruence between the catalyst and the transition state. Molecular model building indicated that a possible substrate was the cyclic phosphate derived from 4-f-butylcatechol (VIII). Indeed, the cyclodextrin bisimidazole (VII) is a catalyst for the cleavage of cyclic phosphate (VIII) (14). 

During last decades the domains C-2 symmetry (the dyad rotation symmetry) of low-B palindrome was established in many enzymes (chymotrypsin, trypsin, aspartyl proteinases, HIV-1 protease, carboxypeptidase A, phospholipase A-2 ribonuclease, etc.) (Lumry, 2002 and references therein). It is proposed that the pair domain closure causes constrain of pretransition state complex that activates cleavage or formation of chemical bonds. Thus control of strong bonds by the cooperation of many matrix or knots bonds takes place. As an example, in the active site of carboxypeptidase A the zinc ion is attached to one of the catalytic domains by histidine 69 and glutamine 72 and connected by hystidine 196 to the second domain. Similar structures were found in the chymotrypsin and pepsin active sites where protons are driven under compression of the domains closure. 

The first determination of the stereochemical course of enzymatic substitution in a phosphate was the study of pancreatic ribonuclease completed by Usher, Richardson and Eckstein and their associates in 1970 [31]. The enzyme had been shown to catalyze the endonucleolytic cleavage of RNA by the two-step catalytic pathway of Equation 11 ... 

The above-mentioned mechanism suggests that positioning the two histidines appropriately would lead to artificial ribonuclease under optimized pH conditions. Figure 6.13 shows an example of an artificial ribonuclease created in this way, which has a cyclodextrin core as the hydrophobic pocket and two histidine residues as catalytic sites. This artificial enzyme catalyzed the second step of the phosphodiester cleavage. The hydrophobic part of the cyclic phosphodiester (substrate) was accommodated into the core of the cyclodextrin and the phosphodiester was exposed between the two histidines. The water molecule was activated through proton removal (performed by the neutral histidine, left), and the activated water performed a nucleophilic attack on the phosphate atom. The protonated histidine (right) assisted this nucleophilic attack by protonating of the phosphodiester. Because of the cooperation between... 

Many enzymes use coenzymes to achieve the detailed transformations they catalyze but the enzyme proteins themselves also supply important elements of the catalysis. Enzyme proteins are the source of the entire catalytic effect when coenzymes are not involved. As one common process, acid and base groups in enzymes perform proton transfers that are critical to the catalytic mechanism. A particularly informative example is observed in the enzyme ribonuclease A, which catalyzes the cleavage of RNA (16). The catalytic process (Fig. 4) involves the imidazole ring of the amino acid histidine that removes the proton from the 2-hydroxyl of the ribose. A different protonated histidine transfers a proton to the RNA to promote the cleavage process. Studies with D2O-H2O mixtures established that the two proton transfers occur at the same time (17). 

Breslow R, Dong SD, Webb Y, Xu R. Further studies on the buffer-catalyzed cleavage and isomerization of uridyluridine. Medium and ionic strength effects on catalysis by morpholine, imidazole, and acetate buffers help clarify the mechanisms involved and their relationship to the mechanism used by the enzyme ribonuclease and by a ribonuclease mimic. J. Am. Chem. Soc. 1996 118 6588-6600. 

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