ENZYMES ATTACKING NUCLEIC ACIDS 593 



adenylic acid group. The phosphomonoester group of TPN, on the other 

 hand, is resistant toward 3'-nucleotidase, and it is assumed that this group 

 is esterified at the 2'-position of the adenyhc acid moiety. The glycerophos- 

 phates and the hexose and pentose phosphates are not hydrolyzed by the 

 enzyme. 



The phosphoric acid diester, diphenyl phosphate, is resistant to 3'-nucleo- 

 tidase, but the phosphodiester linkages of ribopolynucleotides are hydro- 

 lyzed by the enzyme. During this hydrolysis, only up to G% of the total 

 phosphorus is liberated in the form of inorganic phosphate. This would 

 suggest that the interlinkages of the ribopolynucleotides are cleaved at the 

 3'-phosphoric acid ester bonds. It cannot yet be decided whether the ribo- 

 nuclease activity of 3 '-nucleotidase preparations is a property of the 3'-nu- 

 cleotidase itself, or whether it must be attributed to the presence of a 

 second enzyme in the preparations. 



pH Optimum. 3'-Nucleotidase has a rather narrow range of optimal ac- 

 tivity in the neighborhood of p?! 7.5. 



Kinetics. Different 3'-nucleotides are hydrolyzed by 3'-nucleotidase at 

 very different rates. Shuster and Kaplan found the following velocities: 

 3'-adenylic acid, 1; 3'-inosinic acid, 0.47; 3'-guanyHc acid, 0.27; 3'-uridylic 

 acid, 0.19; 3'-cytidylic acid, 0.11. The Km values for some nucleotides are 

 as follows: 3'-adenylic acid, 0.3 X 10"^ M; 3'-cytidylic acid, 2.5 X 10"' M\ 

 3'-uridylic acid, 2.7 X 10-^ M. 



No additive effect on the hydrolysis rates were observed in mixtures of 

 various 3'-nucleotides; this observation suggests the assumption that the 

 hydrolysis of the various 3'-nucleotides is catalyzed by one enzyme. 



The action of 3'-nucleotidase on 3'-nucleotides is competitively inhibited 

 by 2'- and by 5'-nucleotides. 



Inhibition of 50% of the optimal activity is effected by the following 

 compounds: 5.9 X 10-^ M phosphate; 17 X lO"' M arsenate; 0.9 X 10-^ M 

 cysteine; 5.2 X 10~^ M potassium cyanide; 5 X 10~^ M glutathione. The 

 effect of cysteine can be reversed by dialysis. 



Heat Lahility. Most of the enzyme activity is destroyed within 10 min- 

 utes at 80°, and within one or 2 minutes at 100°. 



3. Phosphatases as Phosphotransferases between Phosphoric Acid 

 Esters and Nucleosides 



Brawerman and Chargaff^^'''^-^ obtained evidence for the power of various 

 phosphatases to transfer phosphate groups from "low energy" donors, such 

 as 5'-nucleotides or phenyl phosphates, to nucleosides. This transferase 



i"aG. Brawerman and E. Chargaff, /. Am. Chem. Soc. 75, 2020 (1953). 

 i"b G. Brawerman and E. Chargaff, J. Am. Chem. Soc. 75, 4113 (1953). 

 '^^^ G. Brawerman and E. Chargaff, Federation Proc. 13, 186 (1954); Biochim. el 

 Biophys. Acta (in press). 



