Cysteine ribonuclease

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

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 ... 

Ul. Ubuka, T., Protein disulfide isomerase-catalyzed renaturation of ribonuclease A modified by S-thiolation with glutathione and cysteine. Biochem. Mol. Biol. Int. 38,1103—1110 (1996). 

Fig. 19.2. Amino acid sequence of the enzyme ribonuclease Tj. Cysteine disulfide bridges which cross-link the linear polypeptide chain are indicated by heavy lines [593 a]...

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... 

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]. 

RNase H, wild type E. coli ribonuclease HI with all three free cysteines replaced by alanine, was used in all experiments. RNase H labeled with 35 expressed in E. coli cells grown in M9... 

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... 

A quite different result was obtained when reduced ribonuclease was re-oxidized while it was still in 8 M urea and the preparation was then dialyzed to remove the urea. Ribonuclease reoxidized in this way had only 1 % of the enzymatic activity of the native protein. Why were the outcomes so different when reduced ribonuclease was reoxidized in the presence and absence of urea The reason is that the wrong disulfides formed pairs in urea. There are 105 different ways of pairing eight cysteine molecules to form four disulfides only one of these combinations is enzymatically active. "Fhe 104 wrong pairings have been picturesquely termed scrambled ribonuclease. Anfinsen found that scrambled ribonuclease spontaneously converted into fully active, native ribonuclease when trace amounts of 3-mercaptocthanol... 

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. 

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... 

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