ENZYME KINETICS 



HRP + H2O2 . complex I (1) 



complex I + AH > complex II + A 



complex II + AH » HRP + A 



Here horse-radish peroxidase is represented by HRP, the 

 reduced form of the donor by AH, and the oxidized form by A. 

 Complex I reacts with AH to form complex II at a speed roughly 

 50 times that of the reaction of complex II with AH. Since the 

 conversion of I to II is so rapid, complex II behaves kinetically 

 like the complex postulated by Michaelis. George's titrations 

 of complex II suggested that in the conversion of complex I to 

 complex II, one of the two oxidation equivalents of complex I 

 is transferred to the donor. This was subsequently proved 

 kinetically by Chance. The transient-state data have been 

 correlated point by point with solutions of the rate equations 

 for the above mechanism obtained with the differential analyzer. 



Steady-State Studies 



Most studies of enzyme kinetics have been carried out during 

 the phase of the reaction in which d{ES)/dt is nearly zero. 

 During this phase the velocity drops off as the substrate con- 

 centration decreases. If the initial substrate concentration is 

 used in the rate equation the correct velocity is that obtained 

 by extrapolation to the time of mixing. It should be remembered 

 that the truly initial v^elocity of disappearance of substrate as 

 determined in a transient-state experiment is something entirely 

 different. An alternative to extrapolation of a slowly changing 

 velocity back to zero time is to use an integrated form of the rate 

 equation. This is the basis of the method of Foster and Niemann 

 (6) for determining the maximum velocity and Michaelis con- 

 stant without using initial velocities. 



In order to determine the effects of independent factors such 

 as the concentrations of substrate, hydrogen ions, metal ions, and 

 inhibitors on the steady-state rate, the effect of changing these 

 variables should be studied over as wide a range of concentration 



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