STRUCTURAL BASIS OF RIBONUCLEASE ACTIVITY 



135 



Production of dialyzable nucleotides from RNA by RNase in the presence and 



absence of 8 M urea 



* Incubations contained RNase. 



Fig. 6. Production of dialyzable nucleotides from RNA by RNase in the presence 

 and absence of 8 M urea. 



activity of this enzyme is not dependent on an intact, hydrogen-bonded second- 

 ary Structure. The experiments further suggest that the active center of the 

 molecule may be localized in a very restricted area of the chain. 



Further attack on the problem of function in relation to structure has been 

 made through the study of limited proteolysis of ribonuclease. As mentioned 

 previously, carboxypeptidase treatment does not appear to result in activity 

 loss under circumstances where the C-terminal sequence, -ala.ser.val, is re- 

 moved. 



In another study, carried out by Richards (1955), ribonuclease has been di- 

 gested with the bacterial proteolytic enzyme, subtilisin. From such digests 

 Richards has been able to isolate a modified derivative as shown in Fig. 7, by 

 chromatography on XI-64 resin columns. This derivative, possessing full en- 

 zyme activity, appears to contain one, or possibly more, new N-terminal amino 

 groups. That is to say, subtilisin has cleaved one or more peptide bonds some- 

 where in the interior of the native molecule. The new peptide chain which is 

 formed appears to be attached to the rest of the molecule by a stable cross-link- 

 age. This latter conclusion is based on physical measurements made by Rich- 

 ards on native ribonuclease and on the active derivative following performic 

 acid oxidation. 



Fig. 8 shows, for example, that the sedimentation constant of the oxidized 

 derivative is somewhat lower than that of oxidized native ribonuclease. 



We have recently carried out further proteolytic studies on ribonuclease using 

 pepsin (Aniinsen, 1955). We found several years ago that extremely restricted 

 digestion with pepsin (Aniinsen, 1952) resulted in the complete inactivation of 

 ribonuclease long before the detectable structural changes could be observed. 

 The course of this digestion has now been examined in greater detail, using, once 

 again, XE-64 columns. 



Fig. 9 shows the progressive appearance of an inactive derivative of ribonu- 

 clease at the expense of the native molecule in a pattern of increasing production 



