THE SOLE LYSINE RESIDUE IN PORCINE PEPSIN WORKS AS A KEY RESIDUE FOR CATALYSIS AND CONFORMATIONAL FLEXIBILITY

Pepsin contains a single lysine residue which protrudes from the enzym e's surface, behind the active site cleft, on the C-terminal domain. M utations of pepsin by site-directed mutagenesis of the Lys-319 residue were generated to study the structure-function relationships. Kinetic parameters, pH activity profiles, along with conformational analysis using circular dichroism (CD), and molecular modelling were examined f or the wild-type (non-mutant) and mutant enzymes. The pepsin mutations , Lys-319 --> Met and Lys-319 --> Glu, resulted in a progressive incre ase in the K-m and similar decrease in k(cat), respectively, as well a s being denatured at a lower pH than the wild-type pepsin. CD analysis indicated that mutations at Lys-319 resulted in changes in secondary structure fractions which were reflected in changes in enzymatic activ ity as compared to the wild-type pepsin, i.e. kinetic data and pH dena turation study. Molecular modelling of mutant enzymes indicated differ ences in flexibility in the flap loop region of the active site, the r egion around the entrance of the active site cleft, subsite regions fo r peptide binding, and in the subdomains of the C-terminal domain when compared to the wildtype enzyme. The results suggest that Lys-319, wh ich is distal to the active site, is important to the flexibility/stab ility of the enzyme, as well as to its catalytic machinery.