SPECIFICITY AND INHIBITION OF FUMARASE 



157 



of the enzyme itself. It appears that in the case of fumarase these difficulties 

 may be largely avoided by using buffers of the uncharged base, uncharged acid 

 type, such as /r/5-(hydroxymethyl)-aminomethane acetate, because the ionic 

 composition of the buffer medium may be held constant over a very wide range 

 of pH. 



At low substrate concentrations the initial steady state velocity v for the fu- 

 marase reaction depends upon the substrate concentration according to the 

 ]\Iichaelis equation, 



Vs 

 ' 1 + Ks/{S) 



The maximum initial velocity is represented by Vs and the ^Nlichaelis constant 

 by Ks ■ At high substrate concentrations further terms have to be added to 

 this equation to represent substrate activation and inhibition. The effect of pH 

 on the kinetics of the forward and reverse reactions at low substrate concentra- 

 tions can be summarized by showing the variations of Vs and Ks with pH. Such 

 data are presented in Fig. 1. It is of interest to note that the pH optima for the 

 forward and reverse reactions differ by 1.5 pH units. 



Fumaratp 

 10 mM AcelQte 



pH 



pH 



pH 



Fig. 1. Maximum initial velocities and Michaelis constants for the fumarase re- 

 action at 25° for 0.01 M /m-(hydroxymethyl)-aminomethane acetate buffers (Frieden 

 and Alberty, 1955). The curves have been calculated using equations (3) and (4). 



