ENZYMES AS REAGENTS 



manner that the transfer of an acyl group to an acceptor other 

 than water predominates. The kinetics of an enzyme-catalyzed 

 reaction may also be altered by pH because of a shift to a new 

 rate-limiting Michaelis compound (43). 



SUBSTRATE-ENZYME INTERACTIONS 



In the enzyme-catalyzed reaction: 



E + S . ' ^ ES '-^ E + product 



Km = 



'■m 



1^2 ~r ^3 

 ^1 



the dissociation constant Kj) for the enzyme-substrate complex 

 (ES) is equal to the Michaelis constant iT^ only when ^3 is very 

 small compared to hi', then K^ = k-i/ki = K^. Experimental 

 data are accumulating which demonstrate that Kj) cannot be 

 equated to Kj^. It was shown for some peroxidases {cf. 4,5) and 

 for succinic dehydrogenase (41) that ^3 is considerably larger than 

 k'l, so that Kd may differ from Kj^ by several orders of magnitude. 

 Similar discrepancies have been recorded for enzyme-coenzyme 

 reactions (49). It appears necessary therefore to evaluate the 

 different rate constants independently. Ingenious methods for 

 the direct (4,5) and indirect (41) determination of the velocity 

 constants have been devised. 



In a theoretical paper by Foster and Niemann (8), it was 

 pointed out that in multifunctional catalysis, which involves 

 the formation of several reactive intermediates, the experimental 

 values for kz may not represent the velocity constant for the 

 decomposition of the enzyme-substrate complex. 



Various experimental approaches have been made to the 

 problem of multiple points of substrate attachment. The use of 

 isotopes in the case of symmetrical substrates, the effect of 

 modified substrates, of inhibitors, and of high substrate con- 

 centrations on rate of enzyme activity are among the best known 

 examples. If the enzyme contains two spatially independent 

 points of interaction with the substrate, as shown in Figure 1, 



223 



