N-terminal portion acts as an initiator of the inactivation of pepsin at neutral pH.
Porcine pepsin, an aspartic protease, is unstable at neutral pHs where it rapidly loses activity, however, its zymogen, pepsinogen, is stable at neutral pHs. The difference between the two is the presence of the prosegment in pepsinogen. In this study, possible factors responsible for instability were investigated and included: (i) the distribution of positively charged residues on the surface, (ii) an insertion of a peptide in the C-terminal domain and (iii) the dissociation of the N-terminal fragment of pepsin. Mutations to change the number and the distribution of positive charges on the surface had a minor effect on stability. No effect on stability was observed for the deletion of a peptide from the C-terminal domain. However, mutations on the N-terminal fragment had a major impact on stability. At pH 7.0, the N-fragment mutant was inactivated 5.8 times slower than the wild-type. The introduction of a disulfide bond between the N-terminal fragment and the enzyme body prevented the enzyme from denaturing. The above results showed that the inactivation of pepsin was initiated by the dissociation of the N-fragment and that the sequence of this portion was a major determinant for enzyme stability. Through this study, we have created porcine pepsin with increased pH stability at neutral pHs.
Tanaka
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Department of Food Science, University of Guelph, Guelph,Ontario N1G 2W1, Canada.
Yada
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D004563:Electrochemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010434:Pepsin A / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020116:Pepsinogen A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013552:Swine; D014169:Transformation, Bacterial
D004220:Disulfides; D010446:Peptide Fragments; D020116:Pepsinogen A; D010434:Pepsin A