Improving the alkaline stability of pepsin through rational protein design using renin, an alkaline-stable aspartic protease, as a structural and functional reference.

The present study sought to identify the structural determinants of aspartic protease structural stability and activity at elevated pH. Various hypotheses have been published regarding the features responsible for the unusual alkaline structural stability of renin, however, few structure-function studies have verified these claims. Using pepsin as a model system, and renin as a template for functional and structural alkaline stability, a rational re-design of pepsin was undertaken to identify residues contributing to the alkaline instability of pepsin-like aspartic proteases in regards to both structure and function. We constructed 13 mutants based on this strategy. Among them, mutants D159 L and D60A led to an increase in activity at elevated pH levels (p ≤ 0.05) and E4V and H53F were shown to retain native-like structure at elevated pH (p ≤ 0.05). Previously suggested carboxyl groups Asp, Asp, and Glu were individually shown not to be responsible for the structural instability or lack of activity at neutral pH in pepsin. The importance of the β-barrel to structural stability was highlighted as the majority of the stabilizing residues identified, and 39% of the weakly conserved residues in the N-terminal lobe, were located in β-sheet strands of the barrel. The results of the present study indicate that alkaline stabilization of pepsin will require reduction of electrostatic repulsions and an improved understanding of the role of the hydrogen bonding network of the characteristic β-barrel.