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18] ENOLASE ACTIVITY 339 



no attempts to isolate active fragments have as yet been made. However, 

 with both carboxypeptidasc and aminopcptidase a large number of amino 

 acids can be removed from the enolase molecule without changing the 

 activity. Table 1 summarizes some results on the liberation of amino acids 



Table 1 



THE LIBERATION OF AMINO ACIDS FROM 



ENOLASE ON DIGESTION WITH EXOPEPTIDASES 



AT pH 8-5 AND 22° 



as a function of time of incubation with these two enzymes. In both cases, 

 the molar ratio between enolase and exopeptidase was 30 : 1. The incuba- 

 tions were carried out at pH 8-5; aliquots of the incubation mixtures were 

 taken out at diiferent times and the reaction terminated by precipitating 

 the proteins with trichloroacetic acid to a final concentration of 5 per cent. 

 Amino acids were determined by the ninhydrin method of Moore and 

 Stein,^- and the results, expressed as leucine equivalents, have been cor- 

 rected for small amounts of ninhydrin-positive material Hberated also in 

 the absence of added peptidase. It is apparent that both peptidases liberate 

 close to 100 residues in about 24 hours. In connection with the rather ex- 

 tensive degradation achieved with carboxypeptidasc, it is of interest to note 

 that enolase contains no proline. The removal of this number of amino 

 acids from either the C- or A^-terminal end causes no appreciable loss of 

 activity. The digestion with carboxypeptidasc was found to cause a large 

 change in the electrophoretic mobility of the enzyme. 



The experiments described show that about 17 per cent of the enolase 

 molecule is not essential for maintaining the active site. This is similar to 

 the amount of digestion achieved with ribonuclease without loss of activity,^ 

 but considerably less than in the case of papain,^ where close to 70 per cent 

 of the molecule can be removed. 



While limited removal of the N- and C-terminal chains does not affect 

 the activity, the specific steric configuration of the active site appears de- 

 pendent on non-covalent cross-hnkages, since the enzyme is completely 

 inactive in 8 m urea. This is in contrast to findings with ribonuclease^ but 

 similar to the behavior of many other enzymes. ^^ With enolase, the unfold- 

 ing is reversible during the first fev/ hours of incubation with urea, since the 



