702 



14. EFFECTS OF PH ON ENZYME INHIBITION 



Table 14-3 

 Ionization Constants or Fumarase and Its Complexes with Substrate « 



" From Massey and Alberty (1954) and Frieden and Alberty (1955). Phosphate 

 33.3 mM and acetate 10 mif. 



of the pK^ in the EF complex in acetate, is unexplained. These results are 

 presented to illustrate the magnitudes of the changes that may occur inas- 

 much as no EI complex ionization has been worked out in this detail. 



Negatively charged inhibitors should increase the p/iC^ and i^ositively 

 charged inhibitors should decrease the i^K^. The magnitude of the shift will 

 depend primarily on the amount of inhibitor charge and the distances 

 between the groups. The curve in Fig. 14-14 may be used to estimate the 

 effect, since the ionizing group on the inhibitor will have the same effect 

 on the ionizing group on the protein as will the latter on the former. The 

 shifts observed with fumarase indicate the protein groups to be between 

 6 to 10 A from the carboxylate ions on fumarate and malate (since the charge 

 on these substrates is — 2, the curve in Fig. 14-14 cannot be read off direct- 

 ly, but inasmuch as the shift in piiC,, is proportional to the interaction energy, 

 in this case one-half the observed JpiiT^'s may be used). It is probable that 

 at least one of these groups on fumarase is involved in the catalytic reac- 

 tion and not in the binding (perhaps group b which may be an imidazole). 

 One might expect that charged inhibitors would produce similar effects 

 to those reported for the fumarase substrates, i.e., that a single charged 

 group on the inhibitor that is 8 to 10 A distant from an ionizable group on 

 the enzyme would shift the pE'^ around 0.4 units. Factors other than elec- 

 trostatic may also play a role. An inhibitor molecule might cover or mask 

 an enzyme group so that it would not be readily accessible to protons, or 

 might interfere sterically with it to varying degrees. 



The characterization of the chemical nature of an enzyme group from 

 the experimentally determined p^T^ sometimes presents problems in that 

 it is impossible to predict accurately what the -pK^ of any particular group 

 on an enzyme might be. Ionization constants for some amino acids in dif- 

 ferent states are shown in Table 14-4 and the marked effects of charged 



