Ribonuclease synthetic

The most convincing evidence in favor of a uniform 3,5-diester linkage between nucleotides has been obtained by the action of various enzymes on synthetic diesters of known constitution.218 217 Ribonuclease and spleen extracts were found to act only on nucleoside 3-(benzyl hydrogen phosphates), but not on other isomers, to give nucleoside cyclic phosphates which are broken down further to give nucleoside 3-phosphates. It is concluded, by analogy, that polynucleotides, which are substrates for these enzymes, also possess ester groupings at the 3-positions, rather than at the... 

Of course, the goal of every synthetic organic chemist is to obtain crystalline products, and a few cases of crystalline synthetic proteins have been reported. These include the ribonuclease A synthesized in solution/35 which crystallized after chemical workup and affinity chromatography (see Section 5.1.6.2.2). Further examples include an HIV protease analogue/701 a ubiquitin analogue/59 and monellin/89 which were each prepared by solid-phase methods and purified by HPLC. 

A bacterial peptidase splits a 20-residue fragment containing His 12 from the N-terminal end of RNase A. This "S-peptide" can be recombined with the rest of the molecule, which is inactive, to give a functional enzyme called ribonuclease S. In a similar way, residues 119-124 of RNase A can be removed by digestion with carboxypeptidase to give an inactive protein which lacks His 119. In this case, a synthetic peptide with the sequence of residues 111 -124 of RNase A forms a complex with the shortened enzyme restoring full activity.755... 

With large molecules X-ray crystallographic structure determination may be desirable prior to synthetic activities. Incredibly, ribonuclease, insulin, and a number of smaller peptides have been synthesized. 

The existence of a deoxyribonuclease in E. coli bound to an inhibitory RNA was first suggested by Kozloff (3< ) who found that the DNase activity of freshly prepared extracts could be markedly enhanced by pretreatment with ribonuclease. The enzyme was subsequently purified and freed of inhibitor (39). The purified enzyme termed endonuclease I could, in turn, be competitively inhibited by a variety of RNA s including transfer RNA, and Ri values as low as 10-8 M (nucleotide) have been observed (40). Examination of various purified RNA species and synthetic polyribonucleotides for their inhibitory activity has led... 

It should be pointed out that the successful purification of spleen exonuclease (11) was greatly helped by use of a DNA hydrolyzate produced by spleen acid DNase as the substrate, since the synthetic substrates are nonspecific, and RNA core (the water-undialyzable ribooligonucleo-tides obtained by exhaustive digestion of RNA with pancreatic RNase) is also hydrolyzed by both acid and basic spleen ribonucleases (38, 39). Spleen exonuclease is unable to hydrolyze cyclic phosphates (14). 

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. 

Two other types of specific side-chain interactions have been proposed to stabilize the a helix formed by C-peptide (Shoemaker et al., 1987b). A salt bridge between Glu-2 and Arg-10 has been detected in the crystal structure of ribonuclease A (Wlodawer and Sjolin, 1983) as well as in C-peptide in aqueous solution (Rico et al., 1986). This salt bridge also fixes the N-terminal boundary of the helix between Glu-2 and Thr-3. It is not sterically possible to make this ion pair if Glu-2 is part of the helix. Synthetic studies in which either Glu-2 or Arg-10 is replaced by Ala have provided support for the importance of this interaction in stabilizing the a-... 

Much of the information gleaned from studies of ribonuclease S can be applied to studies of far more complex systems. In most cases the receptor-bound form of the peptide will not be known, but the same synthetic modeling approaches can be applied to probe the conformational features of the complex. Two recent examples of the application of this approach to the design of biologically active peptides will be considered in this section. 

Enzymes which catalyze the hydrolysis of the unit linkage of sequential residues of oligomers or polymers determine their substrate specificity by recognizing the particular unit residue in the sequential chain as well as the direction of the chain. For example, ribonuclease cleaves the 3 -phosphate of a pyrimidine nucleotide residue but not the 5 -phosphate, and trypsin hydrolyzes peptide bonds which involve the arginine or lysine residue at the carbonyl end but not at the amino end. This is also the case for the hydrolysis of a variety of synthetic substrates and quasi-substrates (Sect. 4.1). Synthetic trypsin substrates are ester or amide derivatives in which the site-specific group (positive charge) is contained in their carbonyl portion. 

Chemical synthesis has provided an additional route to peptides containing halogenat-ed amino acids. Early 19F-NMR studies of proteins were performed on semi-synthetic polypeptides prepared by attachment of fluorinated probes to the polypeptide. For example, Heustis and Raftery modified ribonuclease by trifluoroacetylation of Lys residues 1 and 7. They then used 19F-NMR to study conformational changes brought about by the presence of inhibitors200. In his review, Gerig provides several other examples of this strategy187. 

The initial decrease in optical rotation found in aqueous solutions of /3-lactoglobulin and ovalbumin is not, however, sufficient to differentiate globular proteins from simpler synthetic polypeptides in their transition behavior, for neither ribonuclease nor human serum albumin appear to exhibit it. The specific rotation of ribonuclease in water-2-chloroethanol mixtures becomes steadily less levorotatory as the proportion of nonpolar solvent increases (Weber and Tanford, 1959). In the case of human serum albumin Bresler (1958) and Bresler el al. (1959) find that only progre.ssive increases in specific rotation occur as the concentration of 2-chloroethanol is increased and that this change is accompanied by a steady rise in viscosity and the corresponding axial ratios characteristic of the formation of rodlike particles. If these proteins do have some initial helical content in water, as can be argued from their optical rotatory dispersion, then it appears that hydrophobic forces are not required for the stability of these regions. 

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