Contribution of a prosegment lysine residue to the function and structure of porcine pepsinogen A and its active form pepsin A.
A conserved lysine residue, Lys36p, on the prosegment of pepsinogen was replaced with a positively charged arginine (K36pR), a negatively charged glutamic acid (K36pE), and a neutral side chain methionine (K36pM). K36pM and K36pE mutants were extremely unstable and degraded rapidly, especially K36pE, which was inactivated during purification. This instability was confirmed by microcalorimetry where the denaturing temperatures for K36pM and K36pE were 6 degrees C and 10 degrees C lower than the wild-type, respectively. As a function of pH, K36pM and K36pR were activated over a broader range of pH as compared with wild-type. The mutant pepsinogens were activated faster than wild-type with K36pM being activated approximately 10 times faster. The activated pepsins from the various mutant pepsinogens showed lower kinetic efficiency than wild-type enzyme. Catalytic rate constants were reduced by half. The results suggested Lys36p is important for the correct folding of the active-centre residues. The molecular modeling calculation suggested that the position of Asp215 was substantially altered. In conclusion, the above results would suggest that Lys36p was important not only for stability of the prosegment and pepsinogen, but also for the correct alignment of the active-centre residues.
Richter
C
C
Department of Food Science, University of Guelph, Ontario, Canada.
Tanaka
T
T
Koseki
T
T
Yada
R Y
RY
D000818:Animals; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D017931:DNA Primers; D004789:Enzyme Activation; D006868:Hydrolysis; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010434:Pepsin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020116:Pepsinogen A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013552:Swine
D017931:DNA Primers; D020116:Pepsinogen A; D010434:Pepsin A; D008239:Lysine