340 BO G. MALMSTRÖM [18 



activity is regained on dilution. However, after 24 hours in urea solution 

 the inactivation is irreversible. 



The active enolase molecule remaining after proteolytic digestion still 

 contains about 500 amino acid residues, which is much too large to allow 

 structural studies. Thus, as with most other enzymes, information about the 

 active site is of an indirect nature. The influence of pH^^ and solvent com- 

 position^^ on the kinetics and chelating properties implicates the involve- 

 ment of histidine both in metal binding and proton transfer. However, it 

 is hoped that a more direct chemical attack will be possible by utiUzing the 

 strong binding of metal ions to enolase compared with most proteins and 

 peptides. ^^ It is possible that, even after the activity has disappeared, the 

 chelating site is left intact, and attempts to isolate metal-binding fragments 

 after trypsin digestion are now in progress. 



I wish to thank Professor A. Tiselius for his stimulating interest and helpful 

 discussions. Mr. O. Nylander has cooperated in most of the experimental work, 

 and a detailed report will later be published jointly with him. 



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