130 H. GOBIND KHORANA 



very slowly. 33 ' 51, 53 Tobacco mosaic virus ribonucleic acid, in contrast, was 

 found to be phosphorolyzed fairly readily. 51 



(2) Phosphorolysis of Ribo-oligonucleotides. While the macromolecular 

 structure has an important influence on the phosphorolysis reaction, as 

 has further been shown by Grunberg-Manago, 56 ' 56a the studies using well- 

 defined substrates by Singer 57 have shown the requirements of the end 

 group and size for phosphorolysis to occur. Thus, oligonucleotides bearing 

 3'-phosphomonoester or 2',3'-cyclic phosphate end groups are resistant, 

 while oligonucleotides bearing 3 '-hydroxy 1 groups are readily phosphoro- 

 lyzed. The presence of a 5'-phosphomonoester group at the other terminus 

 of the chain is not essential, both series with or without such groups being 

 phosphorolyzed at comparable rates. With equivalent concentrations, a 

 tetranucleotide is more rapidly phosphorolyzed than the corresponding tri- 

 nucleotide. The smallest oligonucleotide susceptible to phosphorolysis is a 

 trinucleotide or a trinucleoside diphosphate, the dinucleotides and dinu- 

 cleoside monophosphates being completely resistant. These results are com- 

 plementary to the findings in the primer work discussed above. The phos- 

 phorolysis reaction has further been shown to be stepwise from the end 

 bearing the 3'-hydroxyl group by a kinetic study of homologous compounds 

 of the type ApApU and ApApApU. In this respect, then, the mode of ac- 

 tion is completely similar 58 to that of the venom diesterase. 59 



The maximal rate of phosphorolysis occurs at 10" 2 M concentration of 

 inorganic phosphate and requires magnesium ions. 



e. Remarks on the Mechanism of Action of Polynucleotide Phosphor ylase 



The studies of Heppel and co-workers, as described above, have estab- 

 lished the stepwise synthesis as well as the phosphorolysis of phosphodi- 

 ester bonds from the 3'-hydroxyl end of the oligonucleotide chains. The 

 reaction, 60 which is depicted below, is formally analogous to the other two 

 enzymic reactions described in later sections, namely, the utilization of a 

 ribonucleoside-5'-triphosphate to attach a nucleoside-5'-phosphoryl group 

 to the 3'-hydroxyl end of a preexisting ribopolynucleotide chain, and like- 

 wise, the addition of deoxyribonucleotide units to deoxyribopoly nucleotide 

 chains. 



Despite the above clarification much remains unknown about the reac- 

 tions catalyzed by polynucleotide phosphorylase (cf., Heppel et al. 56 ). 

 Since none of the enzyme preparations hitherto available have shown an 



67 M. F. Singer, J. Biol. Chem. 232, 211 (1958). 



68 R. J. Hilmoe. Ann. N. Y. Acad. Sci. 81, 660 (1959). 



™ W. E. Razzell and H. G. Khorana, J. Biol. Chem. 234, 2114 (1959). 

 60 Its reversibility indicates that the free energy of hydrolysis of the phosphodiester 

 bond cannot be very different from that of the pyrophosphate bond. 



