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

Abstract

1. The pH-dependence of the binding to pepsin of four dipeptide competitive inhibitors is reported. Values of K(i) 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 K(i) for the corresponding N-acyl-dipeptide acids rise rapidly above pH3.5, and depend on the ionization of a group of apparent pK(a) 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.

Enzyme-mediated Synthesis of Silsesquioxanes

“…6). Inhibition experiments with pepsin were not performed since the classical inhibitors for this enzyme employ solvents, such as ethanol, or acids in order to solubilize the inhibitor molecule(s) [45,46]. The presence of solvents in these reaction systems, especially alcohols, would limit the "green" nature of these reactions.…”

Stability and unfolding studies on alkaline denatured state (Ip) of pepsin

“…The enzyme was found to be pure and used for the present study without further purification. Enzyme concentration was determined spectrophotometrically using an extinction coefficient (e 1% 1cm ) 14.7 [25]. GuHCl was purchased from Sigma Chemicals, USA.…”

Kinetic specificity in papain-catalysed hydrolyses

“…N-Acetyl-L-phenylalanylglycine methyl ester. This was prepared by the method of Knowles, Sharp & Greenwell (1969) Acetyl-L-phenylalanylglycinep-nitrophenylester. Acetyl-L-phenylalanylglycine (0.2g) prepared by the method of Knowles et al (1969) was dissolved in warm ethyl acetate (30 ml).…”

“…This was prepared by the method of Knowles, Sharp & Greenwell (1969) Acetyl-L-phenylalanylglycinep-nitrophenylester. Acetyl-L-phenylalanylglycine (0.2g) prepared by the method of Knowles et al (1969) was dissolved in warm ethyl acetate (30 ml). The solution was cooled to 0°C and p-nitrophenol (0.105g) and NN'-dicyclohexylcarbodi-imide (0.15g) were added successively.…”