Nonspecific Electrostatic Binding of Substrates and Inhibitors to Porcine Pepsin

Abstract

Porcine pepsin has long been recognized to have primary and secondary specificity for hydrophobic amino acids, and the binding of low molecular weight inhibitors (e.g., aliphatic alcohols) is also dominated by hydrophobic interactions¹. However, the long standing ‘hydrophobic dogma’ in pepsin catalysis was seriously challenged when Pohl and Dunn² described a series of polycationic oligopeptides that are among the most reactive synthetic substrates known. These highly hydrophilic molecules should not be well accommodated in a hydrophobic active site. Moreover, the steady state kinetic parameters were remarkably sensitive to the acidity of the medium. As the pH was increased from pH 3 to 6, the specificity number k_cat/K_m increased by three to four orders of magnitude and approached values close to the diffusion limit. The catalytic turnover number k_cat was much less affected by the increase in pH, and the Michaelis constant K_m decreased accordingly, by three to four orders of magnitude. A typical pH profile of steady state kinetic parameters, for the substrate Lys-Lys-Ala-Lys-Phe-Phe(NO₂)-Arg-Leu, is shown in Figure 1.