ENZYME-SUBSTRATE COMPOUNDS 



the actual participants in enzymatic reaction sequences with 

 critical and incisive methods. Direct study of the reaction 

 kinetics of intermediates has been useful for some relatively 

 simple systems (13), and its current application to further com- 

 plexities of single- and multi-enzyme reaction sequences is 

 presented here. 



This paper also illustrates the multiplicity of physical and 

 chemical methods that may be used in the study of complex 

 enzymatic sequences and the use of the analogue computer in 

 order to verify reaction mechanisms that are proposed to 

 represent the enzyme systems. 



One-Enzyme Reaction Sequences 



In the case of catalase and peroxidase the existence of inter- 

 mediates was suggested by over-all studies and was followed 

 by chemical and kinetic studies of the labile intermediate 

 compounds in the reaction sequence. Such studies have given 

 us a definite framework into which the reaction mechanism 

 must be fitted (14,23,36). It is our purpose to examine the 

 theoretical and experimental data on these enzyme reaction 

 mechanisms in order to determine whether undetected inter- 

 mediates precede those already studied, 



CATALASE AND HYDROGEN PEROXIDE 



In the case of catalase, complex I appears to be the primary 

 product of the reaction of catalase and hydrogen peroxide, and 

 is the "Michaelis" enzyme-substrate compound (3). This 

 conclusion is based largely upon the fact that no intermediate 

 compound is observed to precede complex I, even when the 

 reaction is measured in the flow apparatus at very short times 

 (2 millisec). Studies of the over-all reaction of catalase de- 

 scribed below support the idea of direct combination of enzyme 

 and substrate to form complex I in reaction times as short as 

 2 X 10~8 sec. and a similar conclusion is reached on the basis 

 of less extensive data in the case of peroxidase complex I. On 



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