Enzyme ribonuclease

Memfield successfully automated all the steps m solid phase peptide synthesis and computer controlled equipment is now commercially available to perform this synthesis Using an early version of his peptide synthesizer m collaboration with coworker Bemd Gutte Memfield reported the synthesis of the enzyme ribonuclease m 1969 It took them only SIX weeks to perform the 369 reactions and 11 391 steps necessary to assemble the sequence of 124 ammo acids of ribonuclease... 

In many enzymes, the value of kc-JK lies between 108 and 109 M s. The value for L 19 RNA is 103 M 1 s1, i.e., five orders of magnitude lower than for protein enzymes with high catalytic activity. However, L 19 RNA does compare in its efficiency to the enzyme ribonuclease A. The capabilities of ribozymes referred to above dealt solely with interactions of RNA (i.e., ribozymes) with RNA molecules. In a (hypothetical) RNA world, they would, however, need to be capable of doing much more, e.g., carrying out reactions at the carbon skeletons of biomolecules. 

Jokichi Takamine (1854-1922) went to the University of Glasgow, and then, to the United States to investigate the phosphatic manure, and he established the first company of phosphatic manure in Japan. He produced Takadiastase, a digestive agent containing various digestive enzymes, ribonuclease and cellulase. Amylase was extracted from Takadiastase. In a narrow sense, Takadiastase is one of the carboxyoroteases. He discovered in 1900. 

The following reviews describe the molecular and physical properties of this broad class of enzymes that catalyze the endohydrolysis of deoxyribonucleic acids and ribonucleic acids. The class includes deoxyribonuclease II [EC 3.1.22.1], Aspergillus deoxyribonuclease Ki [EC 3.1.22.2], deoxyribonuclease V [EC 3.1.22.3], crossover junction endoribonuclease [EC 3.1.22.4], and deoxyribonuclease X [EC 3.1.22.5]. See also Deoxyribonucleases Restriction Enzymes Ribonucleases... 

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... 

Acid-base titration of the enzyme ribonuclease. The isoionic point is the pH of the pure protein with no ions present except H+ and OH. The isoelectric point is the pH at which the average charge on the protein is 0. [C. I Tanford and J. D. Hauenstein, Hydrogen Ion Equilibria of Ribonuclease." J. Am. Chem. Soc. 1956, 78.5287.]... 

Figure 25-4 Part of amino-acid chromatogram obtained by the method of automatic amino-acid analysis from a hydrolyzed sample of the enzyme ribonuclease. The component amino acids listed are present in the ratio Asp Thr Ser Glu Pro Gly Ala = 15 10 15 12 4 3 12, as determined by peak intensity. The volume of effluent is a measure of the retention time of the amino acids on the column.

In the synthesis of the enzyme ribonuclease by the Merrifield method, the 124 amino acids were arranged in the ribonuclease sequence through 369 reactions and some 12,000 individual operations of the automated peptide-synthesis machine without isolation of any intermediates. 

Ribonucleases are a widely distributed family of en-zymes that hydrolyze RNA by cutting the P—O ester bond attached to a ribose 5 carbon (fig. 8.12). A good representative of the family is the pancreatic enzyme ribonuclease A (RNase A), which is specific for a pyrimidine base (uracil or cytosine) on the 3 side of the phosphate bond that is cleaved. When the amino acid sequence of bovine RNase A was determined in 1960 by Stanford Moore and William Stein, it was the first enzyme and only the second protein to be sequenced. RNase A thus played an important role in the development of ideas about enzymatic catalysis. It was one of the first enzymes to have its three-dimensional structure elucidated by x-ray diffraction and was also the first to be synthesized completely from its amino acids. The synthetic protein proved to be enzymatically indistinguishable from the native enzyme. 

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... 

Enhancement of POD-capacity and appearance of new isoforms is generally considered as an important criterion for senescence (Hazell and Murray, 1982). Lee et al. (1976a) suggested that the cadmium-induced capacity increase of POD and several hydrolytic enzymes (ribonuclease, deoxyribonuclease, acid phosphatase) in Glycine max should constitute an accelerated senescence response. In Zea mays, an induction of leaf acid phosphatase was also reported for toxic concentrations of lead (Maier, 1978 b). 

Cyclodextrin derivatives can act as catalysts, not just as reagents. We are focussing on an attempt to develop a mimic for the enzyme ribonuclease A that incorporates the functional groups of the enzyme, binds an appropriate substrate, and then catalyzes the hydrolysis of such a substrate by a mechanism used by the enzyme itself. Although we want to imitate the mechanism, the selectivity, and the rate of the enzyme, our systems do quite well only with the first two points. They are still quite slow compared with the real enzyme. 

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). 

Enzymes are usually bigger. One of the smaller enzymes—ribonuclease (which hydrolyses RNA) from cows—has a chain of 124 amino acids with four internal disulfide bridges. The abundance of the various amino acids in this enzyme is given in this table. 

The expected trends are born out for the low molecular weight enzymes ribonuclease-a, cytochrome-c, and lysozyme, as shown in Figure 2. These results are presented as the percentage of the protein transferred from a 1 mg/ml aqueous protein solution to an equal volume of isooctane containing 50 mM of the anionic surfactant Aerosol 0T, or AOT (di-2-ethylhexyl sodium sulfosuccinate). As anticipated, only at pH s lower than the pi was there any appreciable solubilisation of a given protein, while above the pi the solubilisation appears to have been totally suppressed. Note, however, that as the pH was lowered even further, there was a drop in the degree of solubilisation of the proteins. This was accompanied by the formation of a precipitate at the interface between the two phases, attributed to a denaturation of the protein. 

Fig. 19.2. Amino acid sequence of the enzyme ribonuclease Tj. Cysteine disulfide bridges which cross-link the linear polypeptide chain are indicated by heavy lines [593 a]...

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). 

Figure 4 The enzyme ribonuclease A cleaves RNA using the imidazole group of histidine 12 as a base and the imidazolium group of histidine 119 as an acid in a simultaneous two-proton transfer process. Studies discussed in this article indicate that the process is not as simple as shown.

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