N-terminal modifications increase the neutral-pH stability of pepsin.
A structure-function study was undertaken to determine the effects of N-terminal mutations in pepsin designed to introduce the Lys-X-Tyr motif and increase N-terminal flexibility. 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. 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, 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 (>or= 4.0) compared to WT, but showed no change for pH dependence of activity, nor a statistically significant change in affinity for the synthetic substrate.
Bryksa
Brian C
BC
Department of Food Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Tanaka
Takuji
T
Yada
Rickey Y
RY
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002384:Catalysis; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020116:Pepsinogen A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013552:Swine; D013912:Threonine / Q000235:genetics
D011993:Recombinant Fusion Proteins; D005973:Glutamine; D013912:Threonine; D018698:Glutamic Acid; D020116:Pepsinogen A; D010434:Pepsin A; D008239:Lysine; D000409:Alanine