VALIDITY OF THE MICHAELIS-MENTEX THEORY 19 



it be assumed, therefore, that one molecule of an enzyme combines with 

 one molecule of a reacting substance, and that the compound molecule 

 exists for a brief interval of time during the further actions which end in 

 disruption and change." The rate-limiting breakdown of this complex was 

 assumed in order to explain the kinetics of /?-fructofuranosidase and it re- 

 mained only for Michaelis and Menten to express this mechanism in mathe- 

 matical form some 10 years later. The history of the ES complex concept 

 is well told by Segal (1959). 



The existence of ES complexes was generally accepted for many years 

 on the basis of the success of the theory, since the demonstration of such a 

 short-lived complex was technically difficult. Direct evidence for the com- 

 plex was first obtained on peroxidase-HgOg by Keilin and Mann (1937). 

 Subsequent spectroscopic work in various laboratories has demonstrated 

 complexes of catalase and HoOg (Chance, 1947), catalase and alkylperoxides 

 (Chance and Herbert, 1950), ribonuclease and ribonucleic acid (RNA) 

 (Hakim, 1957), and aldolase and dihydroxyacetone phosphate (Topper, 

 et al., 1957). The binding of the substrate i\^-acetyl-3.5-dibromo-L-tyrosine 

 to chymotrypsin has been demonstrated by equilibrium dialysis using an 

 exchange reaction with HaO^^ (Doherty and Vaslow, 1952) and a complex 

 between pepsin and ovalbumin was shown by light-scattering measurements 

 at low pH where the hydrolytic rate was slow (Yasnoff and Bull, 1953). 

 Very stable complexes of the acyl derivatives of coenzyme A with acyl 

 dehydrogenases have been isolated (Beinert and Steyn-Parve, 1958; Steyn- 

 Parve and Beinert, 1958). The complexing of DPN+ or DPNH to the follow- 

 ing enzymes has been conclusively demonstrated: alcohol dehydrogenase 

 (Theorell and Bonnichsen, 1951), lactic dehydrogenase (Chance and Neilands, 

 1952), phosphoglyceraldehyde dehydrogenase (Racker and Krimsky, 1952), 

 and DPNH-peroxidase (Dolin, 1956). A number of different types of com- 

 plexes between various dehydrogenases, DPN+, and nucleophilic agents 

 has been observed (Eys et al., 1958). Indirect evidence for complexes is 

 seen in the protection of enzymes from denaturation by their substrates 

 and in the competitive inhibition of many enzymes by structural analogs 

 of their substrates. There would thus seem to be sufficient evidence that 

 the assumption of intermediary complexes is justified but the exact nature 

 of the complex in most instances remains unknown. The assumption of a 

 complex does not specify any particular lifetime, nor does it involve any 

 designated structure for this complex, nor does it restrict in any way the 

 nature of the interaction between enzyme and substrate. 



Assumption That K Is the Dissociation Constant of the Complex 



The interpretation of K as a dissociation constant depends on the assump- 

 tion that the breakdown of the complex into the products is slower than the 

 formation of the complex, limiting the rate of the over-all reaction, and slower 



