Hydrolysis ribonuclease

Oligonucleotides have also been separated by ion-exchange chromatography of yeast ribonucleic acid treated either with acid216 or with ribonuclease.209 Alkaline hydrolysis of the fission products obtained with the latter gives rise to pyrimidine nucleoside 3-phosphates and mixtures of purine nucleoside 2- and 3-phosphates. Bone phosphomonoesterase196 followed by alkaline hydrolysis gives pyrimidine nucleosides and purine... 

Komiyama at al. have prepared two oligonuclear Zn(II) complexes (22 and 23) and tested their hydrolytic activity toward different diribonucleotides [45,46] (catalytic turnover was not demonstrated). The dimer and trimer structures of the active species were confirmed by measuring the hydrolytic activity as a function of Zn/L ratio, which show sharp maxima at the expected 2/1 and 3/1 ratios, respectively. The oligomer complexes have high ribonuclease activity (e.g. the hydrolysis of UpU is accelerated more than 4 and 5 orders of magnitude by 22 and 23, respectively), whereas the effect of the monomer complex 24 was not... 

Vanadium is beneficial and possibly essential for humans. It is certainly essential for a number of organisms. Vanadate (oxidation state V) and its derivatives are phosphate analogues, showing both ground state and transition state analogy (both structural and electronic) with phosphorus compounds. The analogy of five-coordinate vanadium compounds with the transition state of phosphate ester hydrolysis is well documented, and explains why so many vanadium compounds are potent inhibitors of phosphatases, ribonucleases and ATPases. 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

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]. 

Deng et al. (1993,1998) and Ray et al. (1993) have used V " as an analogue of in an attempt to model the transition state of the hydrolysis of phospho-diesters by ribonuclease A since is assumed to adopt the expected five coordination more readily than Examination of the vibrational spectrum of the vanadate analogue indicates that the terminal V-O bonds are only slightly weakened when bonded to the protein. A quantitative bond valence analysis effectively rules out two proposed mechanisms that involve the protonation of the terminal O atoms. 

Fig. 1. The two-step hydrolysis of ribonucleic acid as catalyzed by bovine pancreatic ribonuclease A.

Ribonuclease EC 3.1.27.5 Hydrolysis of RNA Milk is a very rich source similar to pancreatic RNase... 

Ribonuclease, another small globular protein (Mt 13,700), is an enzyme secreted by the pancreas into the small intestine, where it catalyzes the hydrolysis of certain bonds in the ribonucleic acids present in ingested... 

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. 

Sequencing RNA. The first known RNA sequence, that of an alanine tRNA, was determined by Holley and associates in 1965. The RNA was subjected to partial hydrolysis with pancreatic ribonuclease and ribonuclease Tj (Fig. 5-43). Tire small oligonucleotide fragments were separated by ion exchange chromatography under denaturing conditions (7 M urea) and were then characterized individually.645 Tire availability... 

Stereochemical studies support in-line mechanisms for both the transesterification and hydrolysis steps of ribonuclease catalysis. For example, chiral uridine 2, 3 -cyclic phosphorothioates are hydrolyzed with inversion of configuration, with the diastereoisomer shown yielding a 2 -monophosphothioate of the R configuration at phosphorus. 

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),... 

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. 

Rushizky and Sober (82) first described the use of RNase T2 for the base analysis of RNA as it hydrolyzes RNA practically completely to 3 -nucleotides. Ribonuclease T2 digestion is certainly more advantageous for the base analysis of RNA than alkaline hydrolysis which gives... 

Ribonuclease Ua digestion of ApU has revealed reduced hydrolase activity in the second step of RNase U2 action (30). When 87.4% of ApU was readily degraded to produce uridine and adenosine 2, 3 -cyclic phosphate, no 3 -adenylic acid was detected. After exhaustive degradation of ApU, hydrolysis of adenosine 2, 3 -cyclic phosphate occurred and 3 -adenylic acid gradually appeared. 

The first detailed proposal for the mechanism of action of ribonuclease was put forward by Mathias and Rabin and their colleagues (514) An original diagram from their paper is shown in Fig. 28 BIB, 516). It bears a remarkable similarity to the geometry of the active site as defined by the X-ray studies and shown in Fig. 23. For Step 1 the mechanism proposes (1) removal of the proton on the 2 -OH by an imidazole residue in the base form, (2) protonation of the 5 0 of the leaving nucleoside by the other imidazole in the acid form, and (3) attack by the 2 alkoxide on the phosphorus atom to yield the cyclic phosphate. Hydrolysis or alcoholysis of the cyclic phosphate requires the reverse of each of these steps. At the start of step 1, one histidine is in the acidic form and one in the basic form. At the start of step 2 the roles of the two histidine residues are reversed. 

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