The pH-dependence of the binding of competitive inhibitors to pepsin

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Abstract
1. The pH-dependence of the binding to pepsin of four dipeptide competitive inhibitors is reported. Values of Ki obtained from equilibrium-dialysis experiments agree closely with those from kinetic measurements. 2. The binding of uncharged N-acyl-dipeptide amides to pepsin is essentially independent of pH from 0·2 to 5·8. Values of Ki for the corresponding N-acyl-dipeptide acids rise rapidly above pH3·5, and depend on the ionization of a group of apparent pKa 3·6. 3. The data indicate that pepsin does not undergo any gross conformation change (at least none that affects binding) over the whole pH range of its catalytic activity. The pH-dependence of the dipeptide acid inhibitors indicates that the acid anions do not bind to pepsin, presumably because of electrostatic repulsion between the inhibitor anion and a negative centre at or near the active site of the enzyme. 4. The binding of all four stereoisomers of N-acetylphenylalanylphenylalanine, of the depside analogues of the l–l- and d–l-compounds and of N-acetylglycyl-l-phenylalanine and N-acetyl-l-phenylalanylglycine was studied at pH2·2. 5. These results throw further light on the binding specificity of pepsin and on the charge nature of the active site of this enzyme.