ENZYME KINETICS 



The most complete studies of any coenzyme reaction are 

 those by Theorell, Chance, and Bonnichsen (15) on horse liver 

 alcohol dehydrogenase. Since reduced diphosphopyridine 

 nucleotide absorbs strongly at 340 mfx whereas the oxidized form 

 does not, the over-all kinetics were followed spectrophoto- 

 metrically. It was found that when DPNH forms a compound 

 with ADH, the absorption maximum of DPNH shifts from 340 

 to 325 nifx. This observation was the key which opened the door 

 to a detailed study of the stoichiometry, equilibrium, and 

 kinetics. Since this reaction has been studied by both the steady- 

 state and transient-state methods, it is very reassuring to find 

 agreement between the velocity constants calculated from the 

 different types of experiments. It was found that the transient- 

 state kinetic data could be represented by mechanism (10) 

 where E is alcohol dehydrogenase, A is DPNH, C is DPN, B is 

 aldehyde, and D is alcohol. The kinetic data may be interpreted 

 without postulating complexes of aldehyde or alcohol with the 

 enzyme or a ternary complex. Michaelis constants for ethyl 

 alcohol and acetaldehyde are obtained, but this does not mean 

 that these substances necessarily form an intermediate complex 

 with the enzyme. However, data obtained by Fisher et al. (5) 

 using deuterium show that a specific hydrogen atom is trans- 

 ferred from one reactant to the other in the enzymatic reaction. 

 It, therefore, appears that this transfer occurs in a ternary 

 complex of the two reactants with the enzyme, the ternary 

 complex having such a short lifetime that it is not detected 

 kinetically. 



IONIZATION OF THE ENZYME 



Changing the /?H produces large changes in the rates of most 

 enzymatic reactions, even within the range where the enzyme 

 is stable and where the substrate does not change in degree of 

 ionization. Since physiological processes generally occur at a 

 particular p¥L value it is perhaps worth inquiring what can be 

 learned from an investigation of kinetics over a range of /jH 



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