Some observations on the structural basis 

 of enolase activity 



BO G. MALMSTRÖM 



Institute of Biochemistry, Uppsala, Sweden 



Studies on pepsin,^ ribonuclease,'^-* lysozyme,^ and papain^ have demon- 

 strated that the intact protein molecule is not necessary for the catalytic 

 activity of these enzymes. Recent experiments with yeast enolase have shown 

 that this enzyme also can be considerably modified without affecting the 

 enzymic activity. It has been found' that the crystaUine enzyme, prepared 

 according to Warburg and Christian,® contains varying amounts of several 

 active forms (enolase a, b, and c), characterized by different electrophoretic 

 mobilities. The b and c enzymes are beheved to arise from enolase a by 

 proteolytic action during the yeast autolysis [see Malmström'], and this has 

 prompted a study of the effect of limited proteolysis on the activity of 

 enolase. Some preliminary results of these experiments, together with a 

 number of other observations on the structural basis of enolase activity, 

 will be reported in the present communication. 



The enzyme was purified by zone electrophoresis and specific adsorption 

 on to the Mg++ form of a cation exchanger, sulphomethyl cellulose;' this 

 last step takes advantage of the high affinity between enolase and its acti- 

 vating ions. By the new purification procedure it is possible to prepare gram 

 quantities of enolase relatively easily. The method also allows separation 

 of the different active forms of the enzyme, and purified enolase a shows 

 the presence of only one component both in zone electrophoresis and in 

 ultracentrifuge experiments over a wide range of conditions. 



Qualitative end-group studies have been made. Edman's method^ was 

 used for the A^-terminal residues, and DFP (diisopropyl fluorphosphate) 

 treated carboxypeptidase^" for the C-terminal groups. No differences in 

 either N- or C-terminal amino acids could be found between enolase a and 

 by showing that the difference between these forms is not in the end groups. 



The effect of digestion with trypsin, carboxypeptidase and aminopeptidase 

 on the activity of the enzyme has been studied. The trypsin and carboxy- 

 peptidase used were commercial preparations (Worthington), while amino- 

 peptidase was prepared according to Smith and Hill [personal communication 

 (see also Ref.^^)]. Both exopeptidases were treated with DFP prior to use. 

 Trypsin digestion led rather rapidly to a considerable drop in activity, and 



