Understanding the structure–function role of specific catalytic residues in a model food related enzyme: Pepsin

Several aspartic proteinases are used as food processing aids (e.g., pepsin and chymosin in the manufacture of cheese). Aspartic proteinases contain two active site catalytic aspartic acid residues that act as an acid–base pair. Pepsin active site glutamic acid mutants D32E, D215E, and D32E/D215E were compared to wild-type. All mutants were unable to activate autocatalytically, but were able to retain activity and showed decreased turnover numbers (kcat), as well as decreased catalytic efficiencies (kcat/Km). The D32E substitution resulted in a decrease in activity at pH 1–2 suggesting a change in the pKa of the normally conserved residue 32. Circular dichroism revealed that overall secondary structure contents were unaffected by catalytic glutamic acid substitutions whereas thermal stabilities (Tm) were lower relative to wild-type. Energy minimizations predicted that glutamic acid at position 32 altered the positioning of the catalytic carboxyl group more than position 215. This study shows that pepsin can function as a glutamic proteinase.