Engineering cathepsin D-like pH stability into pepsin

This thesis is an investigation of structure-function relationships in pepsin relating to neutral pH stability. Mutant pepsin, E7K/T12A/E13Q, was engineered in an attempt to stabilize the active site by neutralizing net-negative charges by introducing a lysine residue at position 7 into the N-terminus, and two additional mutations at positions 12 and 13 for increased flexibility to allow for rotation of the N-terminus into the active site. Two mutants, E7K and T12A/E13Q, were also studied to investigate the effects of mutations at positions 7, and 12/13, separately. It was demonstrated that at pH 7.0 E7K/T12A/E13Q pepsin was inactivated more slowly compared to WT whereas the mutants E7K and T12A/E13Q were not stabilized. Far-UV circular dichroism revealed that changes in secondary structure accompanied the inactivation process, and that the structural changes occurred at approximately the same rate as inactivation. The above findings support the previously proposed importance of a Lys-X-Tyr motif for stabilization of aspartic proteinase active sites (Lee et al., 1998). All of the inactivated pepsin forms showed retention of substantial secondary structure, more than previously determined for pepsin denatured at pH 7.2 and 8.0, suggesting the presence of a structural intermediate at pH 7.0. The coupled mutations at positions 12 and 13 impacted the pH-dependence of activity at pH 0.9, altered specificity as determined by insulin B-chain cleavage profiles, lowered affinity for a synthetic substrate, and lowered the turnover number. The introduction of Lys at position 7 apparently destabilized the interaction between prosegment-enzyme body as evidenced by activation at higher pH (≥4.0) compared to WT, altered specificity for insulin B-chain, but showed no change for pH-dependence of activity, nor a statistically significant change in affinity for the synthetic substrate.