Ribonucleases amino acids

Christian Anfinsen United States ribonuclease, amino acid sequencing and biological activity ... 

For each entry, a table summarizes information regarding (1) the X-ray diffraction data upon which the analysis is based, (2) the methods employed in structure analysis and refinement and their main results, and (3) some structural characteristics. In a separate table, the ribonuclease amino acid sequence is... 

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

Solid-phase peptide synthesis does not solve all purification problems, however. Even if every coupling step in the ribonuclease synthesis proceeded in 99% yield, the product would be contfflninated with many different peptides containing 123 amino acids, 122 amino acids, and so on. Thus, Menifield and Gutte s six weeks of synthesis was followed by four months spent in purifying the final product. The technique has since been refined to the point that yields at the 99% level and greater are achieved with cunent instrumentation, and thousands of peptides and peptide analogs have been prepared by the solid-phase method. 

FIGURE 5.6 Bovine pancreatic ribonuclease A contains 124 amino acid residues, none of which are tryptophan. Four intrachain disulfide bridges (S—S) form cross-links in this... 

The secondary and tertiary structures of myoglobin and ribonuclease A illustrate the importance of packing in tertiary structures. Secondary structures pack closely to one another and also intercalate with (insert between) extended polypeptide chains. If the sum of the van der Waals volumes of a protein s constituent amino acids is divided by the volume occupied by the protein, packing densities of 0.72 to 0.77 are typically obtained. This means that, even with close packing, approximately 25% of the total volume of a protein is not occupied by protein atoms. Nearly all of this space is in the form of very small cavities. Cavities the size of water molecules or larger do occasionally occur, but they make up only a small fraction of the total protein volume. It is likely that such cavities provide flexibility for proteins and facilitate conformation changes and a wide range of protein dynamics (discussed later). 

William Howard Stein fl 911-1980) was born in New York City and received his Ph.D. in 1938 from the Columbia College of Physicians and Surgeons. He immediately joined the faculty of the Rockefeller Institute, where he remained until his death. In 1972, he shared the Nobel Prize in chemistry for his work with Stanford Moore on developing methods of amino acid analysis and for determining the structure of ribonuclease. 

Fig. 2.—A Portion of the Proton-decoupled, Natural-abundance, 13C-N.m.r. Spectra of Model Compound 6 and Bovine Ribonuclease B at 67.9 MHz. [(A) Compound 8 in HzO (25 mM, pH 6.5) after 8192 scans (2-s recycle-time) (B) spectrum of ribonuclease B after digital subtraction of the spectrum of ribonuclease A. (This enzyme has the same amino acid composition as ribonuclease B, but contains no carbohydrate.) Spectra were taken from Ref. 27.1...

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Despite considerable biochemical work, high-resolution crystal structure determination of native RNase A and S, and some medium-resolution studies of RNase A-inhibitor complexes, a number of questions existed concerning the details of the catalytic mechanism and the role of specific amino acids. Study of the low-temperature kinetics and three-dimensional structures of the significant steps of the ribonuclease reaction was designed to address the following questions. 

Ribonuclease A is a small protein that has 124 amino acids in its single polypeptide chain, molecular mass 13,700. The primary structure of ribonuclease A... 

The fact that a denatured protein can spontaneously return to its native conformation was demonstrated for the first time with ribonuclease, a digestive enzyme (see p. 266) consisting of 124 amino acids. In the native form (top right), there are extensive pleated sheet structures and three a helices. The eight cysteine residues of the protein are forming four disulfide bonds. Residues His-12, Lys-41 and His-119 (pink) are particularly important for catalysis. Together with additional amino acids, they form the enzyme s active center. 

Commercially available computer-automated peptide synthesizers allow synthesis of a polypeptide in a time of about one hour or less per amino acid unit. The ribonuclease synthesis takes about one week or less. 

The S-ribonuclease is the complex formed between an eicosapeptide and the S-RNAse. While replacement of various amino acids by fluorinated analogues does not modify the activity of the native complex, replacement of His-12 by 4-F-His has a strong influence. Indeed, the S-ribonuclease, formed between the bovine pancreatic S-RNAse and the fluoro peptide that contains 4-F-His, has no more catalytic activity, but it is stable. This loss of enzymatic activity is probably due to the significant lowering of the pAia of the catalytic His (2.5 units), which results from the presence of the fluorine atom. It is known that histidine plays an important role in nucleophilic and acid-base processes, which are connected to the catalytic activity of numerous enzymes. 

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