160 



ROBERT A. ALBERTY 



TABLE I 



pK values for groups in fumarase and in fumarase-substrate complexes in 0.01 M 



acetate buffer at 25° 



ionization constants for the groups in tlie free enzyme differ by a factor of 4, 

 which is the statistical factor for a dibasic acid with two independent groups. 



Now, the simple electrostatic effect of bringing a divalent negative ion into 

 the vicinity of an acid group would be to raise the pK value. Malate does this 

 but in the case of fumarate some other effect predominates since one of the pK 

 values is actually reduced. 



Now, why do two ionizable groups exert a total effect on the activity and why, 

 of the three ionized forms of the enzyme-substrate complex, is it the intermedi- 

 ate one which is catalytically active? We believe the answer is that these two 

 groups are actually involved in donating and accepting protons in the catalytic 

 process. This sort of mechanism has been suggested by Nachmansohn and Wil- 

 son (1951) for the cholinesterase reaction and by Swain and Brown (1952) for 

 the catalysis of the mutarotation of tetramethylglucose by o-hydroxypyridine. 



Fig. 3 indicates the kind of mechanism that we imagine for this reaction. Only 

 the step in which the enzyme-fumarate complex is converted into the enzyme- 

 malate complex is shown. The shaded portion represents our ignorance about 

 the structure of the enzyme except for the fact that it contains two groups, R 

 and R', which are involved in the catalytic process. The form of the enzyme- 

 substrate complex which can yield enzyme-product complex is the one in which 



Fig. 3. Mechanism of fumarase action (Alberty, 1956) 



