ENZYMES ATTACKING NUCLEIC ACIDS 565 



The intermediary formation of 2 ',3 '-cyclic pyrimidine nucleotides was 

 suggested by the observation that they could be detected by paper iono- 

 phoresis of ribonuclease digests obtained by very short incubation of 

 ribonucleate with very small quantities of the enzyme. They could be 

 accumulated in considerable ciuantities when the digestion was carried out 

 in dialysis bags so that the cyclic nucleotides were separated from the 

 enzj'me soon after they had been formed. The absence of cyclic nucleotides 

 in later stages of the incubation or in digests obtained with large amounts 

 of ribonuclease is explained by the observation that ribonuclease catalyzes 

 the hydrolysis of cyclic pyrimidine nucleotides to the corresponding 3'- 

 nucleotides. As shown hi Fig. 3, it was concluded that the cleavage of 

 the phosphoryl diester bonds by ribonuclease is not a simple hydroly- 

 sis, but a transphosphorylation (step I) followed by hydrolysis (step 

 II). If A is a pyrimidine nucleotide with a 3 '-phosphoryl group, and B 

 the adjacent nucleotide esterified with this phosphoryl group at the 5 '-pos- 

 ition, the first rapid step of ribonuclease action consists in the shift of the 

 5'-phosphoryl ester bond of B to the 2'-position of A with the formation 

 of a cyclic diester of phosphoric acid (cj^clic nucleotide group or cyclic 

 3 '-nucleotide). The second slow step in ribonuclease action is the spe- 

 cific enzymic hydrolysis of the cyclic diester at the 2 '-phosphoryl ester 

 linkage with the formation of a 3 '-nucleotide group (or nucleotide). 



Nucleotide-P -esters as Substrates of Ribonuclease. Brown and Todd 

 succeeded in synthesizing model esters of nucleotides with benzyl, methyl 

 and ethyl alcohols. It was found that the esters of 3'-cytidine and 3'- 

 uridine phosphates were split by ribonuclease, but that those of the 5'- 

 nucleotides and all esters of adenine nucleotides were resistant to the 

 enzyme. The intermediary formation of cyclic nucleotides was demon- 

 strated during the cleavage of all synthetic nucleotide esters susceptible 

 to the action of the enzyme.^^ 



The behavior of synthetic nucleotide-P-esters of known structure is thus 

 in complete analogy to that of polynucleotides and shows that only one of 

 the two ester bonds of phosphoric acid must be attached to a pyrimidine 

 nucleoside to make the compound susceptible to the action of ribonuclease, 

 and that this bond must be attached to the 3'-hydro.\y group of the ribose 

 moiety. 



Some Synthetic Reactions Catalyzed by Ribonuclease. The conversion of 

 internucleotide bonds to cyclic phosphodiester bonds by ribonuclease raises 

 the c}uestion as to whether or not the reverse reaction might occur bet^^■een 

 cyclic nucleotides and other hydroxy compounds under the influence of 

 this enzyme. Whereas the former reaction results in degradation, the 

 latter would lead to the formation of larger molecules. Model reactions 

 of such a synthetic action of ribonuclease have been demonstrated by 



