Enzyme pancreatic ribonuclease

After three years of work, Holley and his associates managed to collect 1 g of alanine transfer RNA, which they used to study the sequence of the polynucleotide. To perform this task, investigators used three different enzymes pancreatic ribonuclease, taka-diastase (Tl), and snake venom phosphodiesterase. 

Fig. 7. Quantity of enzymes (m) ]) terrilytin and 2) pancreatic ribonuclease, bonded by crosslinked MA-DMEG copolymers with different contents of the crosslinking agent...

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. 

Papain is readily inactivated by PAN (at 115 ppm for 40 min), provided that it is in the sulfhydryl form. The reaction of sulfhydryl groups of hemoglobin with PAN is very similar to the reaction with p-mercuricbenzoate there is more reaction at a pH of 4.5 than at a pH of 7. However, there is one striking difference between PAN and classic compounds that react with sulfhydryl groups egg albumen is resistant to reaction with PAN. Thus, enzymes that have no free sulfhydryl groups should be quite resistant to PAN. This is the case with pancreatic ribonuclease the native enzyme was not affected by a 300-fold molar excess of PAN. 

Many secretory proteins—e. g., pancreatic ribonuclease (RNAse see p. 74)—contain several disulfide bonds that are only formed oxidatively from SH groups after translation. The eight cysteine residues of the RNAse can in principle form 105 different pairings, but only the combination of the four disulfide bonds shown on p. 75 provides active enzyme. Incorrect pairings can block further folding or lead to unstable or insoluble conformations. The enzyme protein disulfide iso-merase [1] accelerates the equilibration between paired and unpaired cysteine residues, so that incorrect pairs can be quickly split before the protein finds its final conformation. 

The question of enzyme specificity for irradiated polynucleotides is taken up in more detail in the recent review of Johns.11 The specificities of four enzymes, spleen phosphodiesterase, snake venom phosphodiesterase, pancreatic ribonuclease, and pancreatic deoxyribonuclease are discussed. 

A series of carboxyl derivatized polyglucoses were studied as inhibitors of ribonuclease activity, in an attempt to relate charge density to inhibitory activity.202 In comparison with other factors, it was concluded that coulombic forces probably play a major role in complex-formation between enzyme and substrate, and between enzyme and inhibitor. However, other specific, nonelectrostatic forces were shown to participate in the binding of bovine pancreatic ribonuclease to ribonucleic acid.204... 

RIBONUCLEASE. An enzyme that causes splitting of ribonucleic acid, Pancreatic ribonuclease for example, cleaves only phosphodiester bonds... 

In most species the level of pancreatic ribonuclease is quite low. Its function presumably is the digestion of exogenous RNA in the diet. In ruminants there is very much more of the enzyme, and Barnard (12) has concluded that the primary purpose of pancreatic ribonuclease is digestion of the RNA of the bacteria in the rumen rather than of the dietary RNA. The reutilization of the nitrogen and phosphorus of this... 

In a classic study on bovine pancreatic ribonuclease A at 90°C and pH conditions relevant for catalysis, irreversible deactivation behavior was found to be a function of pH (Zale, 1986) at pH 4, enzyme inactivation is caused mainly by hydrolysis of peptide bonds at aspartic acid residues as well as deamidation of asparagine and/or glutamine residues, whereas at pH 6-8, enzyme inactivation is caused mainly by thiol-disulfide interchange but also by fi-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. 

This process is routinely automated in commercially available machines. Solutions of all of the protected amino acids required are stored in separate containers and a programmed sequence of coupling and deprotection leads rapidly to the complete peptide in days rather than the years needed for solution chemistry. The most dramatic illustration of this came with the publication of a heroic traditional synthesis of bovine pancreatic ribonuclease A (an enzyme with 124 amino acids) by Hirschmann, side-by-side with one by Merrifield using functionalized polystyrene as we have described. The traditional method required 22 co-workers, while the Merrifield method needed only one. 

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 ... 

Two potentially cytotoxic proteins were isolated in this manner, bovine pancreatic ribonuclease A (RNase A) and a restriction enzyme from Haemophilus parainfluenzae (Hpa ) (55). A naturally occurring cysteine residue close to the... 

Richards FM, Wycoff HW. Bovine Pancreatic Ribonuclease. The Enzymes, Volume 4. 3rd edition. 1971. Academic Press, New York. pp. 647-806. 

Blackburn, P. and Moore, S. (1982). Pancreatic ribonuclease. The Enzymes, New York, Academic. 317-433. 

Bovine pancreatic ribonuclease (RNase) catalyses the hydrolysis of RNA by a two-step process in which a phosphate intermediate is formed (Richards and Wyckoff, 1971). The enzyme consists of a single polypeptide chain of molecular weight of 13.7 kD. The bond between Ala-20 and Ser-21 may be cleaved by subtilisin, but the resulting peptide... 

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