Lysine residues ribonuclease

Brown LR, Bradbury JH. Proton-magnetic-resonance studies of the lysine residues of ribonuclease A. Eur. J. Biochem. 1975 54 219-227. 

The structures of the native enzyme and its complexes with several inhibitors have since been obtained at higher resolution in other laboratories, to afford a more complete description of the enzyme - substrate interactions.191 Particularly noteworthy are the lysine residues 7,41, and 66. That these are an important part of the catalytic machinery has been deduced from their conservation in evolution (they have been found in all homologous ribonucleases that have been sequenced), and from their loss of activity when they are acetylated. Lys-41 is particularly important. The lysine side chains are very mobile in the free enzyme, but their mobilities are much decreased on the binding of nucleotide substrate analogues. Lys-41 interacts directly with the phosphate moiety and is thought to stabilize the pentacovalent intermediate. Another residue that has been con-... 

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. 

However, the specific activities of certain amino acid residues are significantly different when the two reagents are compared. Thus, the specific activity of lysine in ribonuclease labeled with TI is lower and that of alanine is higher than for labeling with HST. It appears, therefore, that some distortion can occur when tritiated scavengers are employed. It was pointed out earlier that a possible explanation of such distortions might lie in the reactions by which the radicals produced... 

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. 

Ribonuclease A.—Lactose has been coupled to L-lysine residues in the cross-linked dimer of bovine pancreatic ribonuclease A by reductive amination with cyanoborohydride. Derivatives of the dimer containing up to ten deoxy-lactitoyl)-L-lysine residues per molecule retained more than 75% of the activity of the native enzyme. The effect of modification of the enzyme on hepatic uptake was investigated. 

The pH-rate profile for the action of the enzyme shows a typical pH maximum, with sharply lower rates at either higher or lower pH than the optimum these facts suggest that both an acidic and a basic group are required for activity (Herries, 1960). The two essential histidine residues could serve as these groups if, in the active site, one were protonated and the other present in its basic form. The simultaneous acid-base catalysis would parallel that of the model system (discussed below) of Swain and J. F. Brown. The essential lysine, which binds phosphate, presumably serves to bind a phosphate residue of the ribonucleic acid. These facts led Mathias and coworkers to propose the mechanism for the action of ribonuclease that is shown in (13) (Findlay et al., 1961). 

Ribonuclease A hydrolyzes RNA adjacent to pyrimidine bases. The reaction proceeds through a 2, 3 -phosphate cyclic diester intermediate. Formation and breakdown of the cyclic diester appear to be promoted by concerted general-base and general-acid catalysis by two histidine residues, and by electrostatic interactions with two lysines. These reactions proceed through pentavalent phosphoryl intermediates. The geometry of these intermediates resembles the geometry of vanadate compounds that act as inhibitors of the enzyme. 

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

Each protein has, within this general pattern, its own characteristic radical distribution. In ribonuclease, lysine exhibits a much higher activity than do the remaining amino acids. Lysine and methionine are the most heavily labeled residues in lysozyme. In myoglobin, histidine has the highest activity. Methionine is the most heavily labeled amino acid in chymotrypsinogen, as is proline in insulin. Despite the similarities, therefore, each native protein exhibits a characteristic tritium distribution. 

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