ENZYMES ATTACKING NUCLEIC ACIDS 563 



field and Woollej',-^ who succeeded in isolating by ion-exchange chromatog- 

 raphy a considerable number of di- and trinucleotides from hydrolysates 

 of yeast ribonucleic acid obtained by short treatment of PNA with 6 N 

 hydrochloric acid at room temperature. All those oligonucleotides in which 

 the 3'-phosphoryl groups of pyrimidine nucleotides were esterified with 

 other nucleotides were cleaved by ribonuclease, whereas all oligonucleo- 

 tides in which such a structure was absent were resistant toward the 

 enzyme. 



The presence of pyrimidine residues in those nucleotide groups which 

 react with ribonuclease is, however, not a specific recjuirement for the 

 catalytic action of the enzyme. Zamenhof et al}- isolated from Hemophilus 

 influenzae, type b, a polymer of ribosyl-3-phosphoric acid which was hydro- 

 lyzed by ribonuclease (Fig. 2). It would appear, then, that the contrast in 

 the behavior of pyrimidine and purine nucleotide groups against ribonu- 

 clease must be interpreted as a specific resistance of the purine nucleotide 

 groups against the action of ribonuclease rather than by a specific impor- 

 tance of the pyrimidine group for the catalytic activity of this enzyme. 



Mechanism of Action of Ribonuclease (see also Chapter 12). The enzymic 

 cleavage of internucleotide bonds by ribonuclease is not a simple hydrolysis 

 but an intramolecular transphosphorylation followed by hydrolysis. The 

 presence of transphosphorylating and hydrolyzing activities in the same 

 enzyme is not a unique property of ribonuclease, but has recently been 

 observed in phosphomonoesterases. However, transphosphorylation as a 

 necessary intermediary step of hydrolysis on the substrate level has so 

 far been established only for the action of ribonuclease on polynucleotides.- 



The concept of the biphasic nature of the cleavage of ribonucleic acids 

 by ribonuclease is based on the following observations. Chantrenne, Linder- 

 str0m-Lang, and Vandendriessche'^"-^ made the interesting discovery that 

 the volume changes of a ribonucleate solution during the action of ribo- 

 nuclease were biphasic: A short and transitory dilatation of the solution 

 was followed by a slow contraction. Since the simple hydrolytic clea\age 

 of an ester bond would be expected to result exclusively in a small con- 

 traction, these authors concluded that the cleavage of the internucleotide 

 bonds of ribonucleic acid by ribonuclease was not a simple hydrolysis, but 

 that it followed a more complex mechanism. 



2' R. B. Merrifield and D. W. Woolley, J. Biol. Chem. 197, 521 (1952). 



"^ As contrasted with hypothetical transphosphorylation between an "active" en- 

 zyme phosphate compound and the substrate. 



*' H. Chantrenne, K. Linderstr0m-Lang, and L. Vandendriessche, Nature 159, 877 

 (1947). 



^* L. Vandendriessche, Conipt. rend. trav. lab. Carlsberg, Ser. chim. 27, 341 (1951). 



-^ L. Vandendriessche, Acta Chem. Scand. 7, 699 (1953). 



