Ribonuclease-S and

The methionine 29 is on the outside of ribonuclease-S and simple absorbed [PtClJ2- or [PtC en] should have been rapidly removed from this site. We therefore believe that the platinum complex has reacted... 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

In a more recent study using circular dichroism, Pflumm and Beychok (313) have fitted the observed curve for RNase-A (see Figs. 11c and d) weighted mixtures of the characteristic bands for helix from poly-L-glutamic acid and / structure from poly-L-lysine. The data are compatible with 11.5% helix and 33% / conformation. Ribonuclease-S and RNase-A have almost identical CD spectra from 198 to 300 nm. The spectrum of S-protein is markedly different from the other two. 

Maruyama, T., Sonokawa, S. and Matsushitaa, H. Goto, M. (2007) Inhibitory effects of gold(III) ions on ribonuclease and deoxyribonuclease. Journal of Inorganic Biochemistry, 101, 180-186. 

Simonson, T. Brimger, A. T., Thermodynamics of protein-peptide binding in the ribonuclease S system studied by molecular dynamics and free energy calculations.,... 

Vithayathil, P.J., and Richards, F.M. (1960) Modification of the methionine residue in the peptide component of ribonuclease-S./. Biol. Chem. 235, 2343-2351. 

Fig. 1. Schematic drawing of the polypeptide backbone of ribonuclease S (bovine pancreatic ribonuclease A cleaved by subtilisin between residues 20 and 21). Spiral ribbons represent a-helices and arrows represent strands of /3 sheet. The S peptide (residues 1-20) runs down across the back of the structure.

Fig. 33. Stereo drawings of particular examples of types Via (a) and VIb (b) cis-proline turns, (a) Ribonuclease S 91-94 (Lys-Tyr-Pro-Asn) (b) Bence-Jones protein REI 6-9 (Gln-Ser-Pro-Ser).

One simple case of disordered structure involves many of the long charged side chains exposed to solvent, particularly lysines. For example, 16 of the 19 lysines in myoglobin are listed as uncertain past C8 and 5 of them for all atoms past C/J (Watson, 1969) for ribonuclease S Wyckoff et al. (1970) report 6 of the 10 lysine side chains in zero electron density in trypsin the ends of 9 of the 13 lysines refined to the maximum allowed temperature factor of 40 (R. Stroud and J. Chambers, personal communication) and in rubredoxin refined at 1.2 A resolution the average temperature factor for the last 4 atoms in the side chain is 9.2 for one of the four lysines versus 43.6, 74.4, and 79.3 for the others. Figure 57 shows the refined electron density for the well-ordered lysine and for the best of the disordered ones in ru-... 

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. 

The solid-phase method was tested with the synthesis of many small peptides,[19] including bradykinin, angiotensin, desaminooxytocin, and the 20-residue S-peptide of ribonuclease A, and was found to be useful and efficient. 

The five N-terminal residues and the six or seven C-terminal residues cannot be seen in the high resolution electron density map, and the loop referred to above, formed by residues 44 to 53, appears at only one-third to one-half the amplitude of the well-resolved parts of the map. The lack of clarity in these three regions might possibly result from poor phasing or some other crystallographic factor, but we consider it more likely that these predominantly hydrophilic sections of the peptide project in a disordered way into the solvent. In this connection, it is interesting that in the presence of Ca2+ and pdTp trypsin cleaves inhibited nuclease at only two points between residues 5 and 6 and between residues 48 and 49 (36-38) which are at the very extremity of the loop. It also seems relevant that ribonuclease S also shows lack of clarity at the ends of the peptide chains and in the region of a relatively exposed loop (56). 

Ribonuclease-S can be separated into S-peptide [residues 1-20 (21)] and S-protein [residues 21 (22)-124] by precipitation with trichloroacetic acid 73) or better, Sephadex chromatography in 5% formic acid 83). The best preparations of these components show no detectable hydrolytic enzymic activity and little if any transphosphorylation activity (see Section VI). Isolated S-peptide appears to have no regular secondary structure 83, 84) or 10-20% helicity 85, 86). (These slightly different interpretations are based on almost identical CD data.) When equimolar amounts of S-protein and S-peptide are mixed at neutral pH and room temperature or below, essentially full catalytic activity is recovered 73, 87). A schematic diagram is shown in Fig. 7. For a detailed summary of the preparative procedures see Doscher 88). 

Perturbation of the spectrum of RNase-A by dioxane was almost exactly one-half that of Ox-RNase or of that expected for 6 free tyrosyl residues (303). This solvent was not included in the series reported by Herskovits and Laskowski (301). The results appear to indicate a 3 3 division but could, of course, also fit the 2 2 2 division if the appropriate characteristics are attributed to the free, partially accessible, and buried groups. Ribonuclease-S also appears to have 3 buried tyrosyl residues and S-protein only 2 (304). Presumably Tyr 25 is normalized when S-peptide, and thus the Asp 14 interaction, is removed. 

Fig. 11. (a) Far ultraviolet rotatory dispersion of ribonuclease. Corrected mean residue specific rotation vs. wavelength [to R = [aLAf/100 [3/(n2 + 2)l where a — specific rotation, M mean residue weight, and n = solvent refractive index. Bars give maximal deviation at peaks. Reproduced from Jirgensons (311). (b) Near ultraviolet rotatory dispersion of 0.48% pancreatic ribonuclease in a 1-mm cell, in (a) 0.15 M phosphate buffer at pH 62 (b) 0.15 M glycine-NaOH buffer at pH 11.5 (c) 0.1 N HC1 (d) 15% sodium dodecyl sulfate. Reproduced from Glazer and Simmons (313). (c) Far ultraviolet circular dichroic spectra of RNase-A, RNase-S, and S-protein at 25° and 3°. Reproduced from Pflumm and Beychok (313). (d) Near ultraviolet circular dichroic spectra of RNase-A as a function of pH. Reproduced from Pflumm and Beychok (313). 

Shlesinger, P.H., Doebber, T.W., Mandell, B.F., White, R., DeSchryver, C., Rodman, J.S., Miller, M.J. and Stahl, P.D. (1978) Plasma clearance of glycoproteins with terminal mannose and W-acetylglucosamine by liver non-parenchymal cells. Study with betaglucuronidase, N-acetyl-beta-D-glucosamine, ribonuclease B and agalacto-orosomucoid.Biochem. J., 176, 103-108. 

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