ENZYME KINETICS 



intermediate. Actually it turns out to be impossible from 

 kinetic data alone to specify the composition of such an inter- 

 mediate. However, the fact that the rate of appearance of 

 intermediate is directly proportional to the concentration of sub- 

 strate and free enzyme proves that the activated complex for the 

 reaction contains one substrate molecule and one enzyme mole- 

 cule. The product of this second-order reaction may be some- 

 thing less than a complex containing the whole of both molecules. 

 One of the themes of this article is that the basic mech- 

 anism of Michaelis and Menten can be extended and enlarged 

 upon in many ways to include further phenomena while retain- 

 ing the essential novel idea of the reversible formation of an 

 intermediate by substrate and enzyme which can decompose or 

 react to yield product and regenerate the enzyme. 



Transient-State Studies 



In general, the following three stages may be distinguished 

 in a simple enzymatic reaction. First, immediately after mixing 

 solutions of enzyme and substrate, there is a disappearance of 

 substrate and free enzyme as they react with each other. For 

 most enzymatic reactions this process is so fast that it has been 

 impossible to study it. As the concentration of enzyme-substrate 

 complex increases the rate of appearance of product increases 

 until at a particular concentration of enzyme-substrate complex 

 there is a steady state in which the rate of formation of the 

 complex is balanced by its rate of decomposition. In this second 

 stage of the reaction the rate of appearance of product is con- 

 stant. If the substrate concentration is high the reaction velocity 

 may remain constant for a sufficiently long time to permit its 

 accurate determination. On the other hand, if the concentration 

 of substrate is low, the reaction rate may drop rapidly as the 

 substrate is used up. In this third and final stage of the reaction, 

 the concentration of the enzyme-substrate complex decreases. 

 Studies of all three phases of the reaction give the most complete 

 information about the mechanism and are referred to as tran- 

 sient-state studies. Chance (3), who in 1940 first studied the 



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