Ribonuclease functional groups

The essential—SH group in D-glyceraldehyde-3-phosphate dehydrogenase and the imidazol residues of the ribonuclease are also more reactive because of side-chain interactions in the active center. Such functional groups may have such extremely high reactivity that an equivalent amount of the reagent causes full inactivation of the enzyme. 

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. 

Below is part of the structure of ribonuclease surrounding one of the catalytic amino acids Hisl2. There are seven amino acids in this sequence. Every one is different and every one has a functionalized side chain. This is part of a run of ten amino acids between Phe8 and Alai 9. This strip of peptide has six different functional groups (two acids, one each of amide, guanidine, imidazole, sulfide, and alcohol) available for chemical reactions. Only the histidine is actually used. 

Preparations of pancreatic ribonuclease, ribonuclease from Actinomyces rimosus, and the nuclease from Serratia marcescens have been covalently bound to 4 3-hydroxyethylsulphonylanisole-, 4j3-hydroxyethylsulphonylaniline, and 3-Cl-2-hydroxypropyl derivatives of dextran and dialdehyde-dextran and to the 4-aminobenzyloxymethyl ester of dextran.The yield and thermal stability of the modified nucleases depends both on the amount and the character of the functional groups activating the polysaccharide. 

In contrast, completely artificial ribonucleases are designed and synthesized according to the functions and properties we need, and thus factors (l)-(3) can be minimized. Furthermore, even non-natural functional groups can be easily incorporated in these man-made catalysts. 

Group-specific reagents there are a variety of reagents which can specifically modify functional groups found in enzymes, e.g. iodoacetate at pH 5.5 was employed to identify specific histidine residues important in the catalytic activity of pancreatic ribonuclease. The other histidine residues in this enzyme molecule are less reactive with iodoacetate. 

Breslow s P-cyclodextrine ribonuclease model system represents one of the best examples concerning the construction of small enzyme-like molecules [33]. Breslow functionalized the P-cyclodextrine with two imidazole moieties (Figure 10.1). Selectively, catechol cyclic phosphate carrying a 4-tert-butyl group (Figure 10.1a) binds into the cavity of the catalyst (Figure 10. lb) in water solution, and is then hydrolyzed by the... 

Ribonuclease is an enzyme with 124 amino acids. Its function is to cleave ribonucleic acid (RNA) into small fragments. A solution containing pure protein, with no other ions present except H+ and OH- derived from the protein and water, is said to be isoionic. From this point near pH 9.6 in the graph, the protein can be titrated with acid or base. Of the 124 amino acids, 16 can be protonated by acid and 20 can lose protons to added base. From the shape of the titration curve, it is possible to deduce the approximate pATa for each titratable group.1-2 This information provides insight into the environment of that amino acid in the protein. In ribonuclease, three tyrosine residues have "normal values of pATa(=10) (Table 10-1) and three others have pA a >12. The interpretation is that three tyrosine groups are accessible to OH, and three are buried inside the protein where they cannot be easily titrated. The solid line in the illustration is calculated from pA"a values for all titratable groups. 

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