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 enzyme's surface, behind the active site cleft, on the C-terminal domain. Mutations 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 for the wild-type (non-mutant) and mutant enzymes. The pepsin mutations, Lys-319-->Met and Lys-319-->Glu, resulted in a progressive increase in the Km and similar decrease in kcat, respectively, as well as 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 activity as compared to the wild-type pepsin, i.e. kinetic data and pH denaturation study. Molecular modelling of mutant enzymes indicated differences in flexibility in the flap loop region of the active site, the region around the entrance of the active site cleft, sub-site regions for peptide binding, and in the subdomains of the C-terminal domain when compared to the wild-type enzyme. The results suggest that Lys-319, which is distal to the active site, is important to the flexibility/stability of the enzyme, as well as to its catalytic machinery.
Cottrell
T J
TJ
Department of Food Science, University of Guelph, Ontario, Canada.
Harris
L J
LJ
Tanaka
T
T
Yada
R Y
RY
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry; D010434:Pepsin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010435:Pepsinogens / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
D017931:DNA Primers; D009842:Oligopeptides; D010435:Pepsinogens; D010434:Pepsin A; D008239:Lysine