Cysteine residues ribonuclease

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. 

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. 

Oxidation of mono-cysteine peptides to the dimer is a straightforward reaction that can produce only the desired product. In the case of bis-cysteine peptides statistically the oxidation leads to the homodimers in parallel and antiparallel orientation as well as to the disulfide-bridged monomer and oligomers. When the two cysteine residues are placed in the adjacent position formation of homodimers is highly favored over the cyclic monomer (Section 6.1.5.1) and the product distribution depends strongly on the peptide concentration. Such a type of intermolecular disulfide bridging is present in bovine seminal ribonuclease, where an antiparallel alignment occurs at the interface of the dimer. 97 ... 

Fig. 12. Periodicity of occurrence of lysine and cysteine residues in ribonuclease. 

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... 

Deprotection of peptides. In a synthesis of bovine pancreatic ribonuclease, the 33 protective groups required were removed by three treatments with 1 M TFMSA-thioanisole at 0° for 60 minutes. The groups were Cbz, r-butyl, p-methoxybenzyl, and p-methoxybenzenesulfonyl.1 The final step involved air oxidation of the cysteine residues to disulfides mediated by glutathione.2... 

Formation of disulfide bonds from cysteine residues is an oxidation reaction. A cysteine residue in a-amylase is oxidized at pH 8.O.818 Methionine and histidine residues are also susceptible to oxidation. Oxidation of methionine residues has been observed during storage of parathyroid hormone819 and relaxin.820 Degradation of freeze-dried ribonuclease A was ascribed to oxidation because molecular oxygen was involved in the degradation process.821... 

Structure governing the ionization or pK of cysteine residue will also influence the cysteine oxidation rate [49, 59], As demonstrated using seminal ribonucleases segment 29-34 [60] and a-lactalbumin [61], introduction of an electronegative environment into the structures raises the pK of the thiol groups and reduces the cysteine oxidation rate. In contrast, cysteine residue in papain, which displays a lower pKa due to the presence of a nearby ionizable histidine group, is chemically more reactive than expected [59],... 

Parente A, Merrifield B, Geraci G, et al. (1985). Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonucleases. Biochem. 24 1098-1104. 

The reaction of hexahydropyrroloindole (HPI) (46) with thiols to give the corresponding 2-thioether-tryptophan compounds has been further investigated (464). Reaction of cysteine with HPI (1.2 equiv) in 25% tri-fluoroacetic acid produces quantitatively tryptathionine, an amino acid contained in the toxic peptides of Amanita phalloides (see Section VI.2.4.). Reduced ribonuclease, a protein containing 8 cysteine residues per molecule, was treated with HPI, and the modified protein purified by gel filtration. The completeness of the reaction was confirmed by hydrolysis with /7-toluenesulfonic acid (233) and analysis of the hydrolyzate. A value of 7.6 (theory 8) residues per mole of protein of oxindolylalanine, the product of hydrolysis of the tryptathionine residues (431) (see Section III.4.2.), was obtained. This new reaction of cysteine residues should be of value in peptide synthesis, providing a simple method for linking tryptophan and cysteine as a basic step in the chemical synthesis of the peptides of Amanita phalloides. 

Luse and M(iLaren (1963) have reviewed published research on the photolysis products and quantum yields tor the destruction of amino acids and have attributed the photochemical inactivation of the enzymes chymo-trypsin, lysozyme, ribonuclease, and trypsin by UV light at 254 m i primarily to destruction of the cystyl and tryptophyl residues. The destruction of these residues in proteins was suggested to be a function of the product of the number of residues present, the molecular extinction coefficient, and the quantum yield for destruction of each residue. Cysteine and tryptamine were identified among the irradiation products from cystine and tryptophan, respectively. Tyrosine, histidine, and phenylalanine were also shown to be degraded by UV, histidine yielding histamine, urocanic acid, and other imidazole derivatives, and phenylalanine yielding tyrosine and dihydroxyphenylalanine. Destruction of these three amino acids was not considered to contribute appreciably to the enzyme inactivation. 

The S-Me methionine H resonances are shifted and broadened when PtCli binds to ribonuclease [117] (Figure 3.8). In its native state, only one residue is accessible, but all four become available for binding at low pH. Cd and Zn binding to metallothionein (mol. wt. 12000) begins at pH 2, and cysteine methylene H resonances are broadened [118]. 

The Merck3 total synthesis of ribonuclease involved coupling various peptide units to form a tetrahectapeptide (104 residues) known as S-protein. This polypeptide contains 8 cysteine and 3 methionine residues. The acetamidomethyl blocking group proved invaluable for protection of the cysteine units. 

The cited evidence for the B-elimination mechanism leading to dehydroalanine formation merits further comment. Nashef et al. (41) report that alkali-treatment of lysozyme ribonuclease and several other proteins resulted in loss of cystine and lysine residues and the appearance of new amino acids lysinoalanine, lanthionine, and B-aminoalanine. Alkali-treatment of the proteins induced an increase in absorbance at 241 nm, presumably from the formation of dehydroalanine residues. The dehydroalanine side chain can participate in nucleophilic addition reactions with the e-NH2 group of lysine to form lysinoalanine, with the SH groups of cysteine to form lanthionine, and with ammonia to form B-aminoalanine. 

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