Truncated prolyl oligopeptidase from Pyrococcus furiosus.
The peptidase domain of prolyl oligopeptidase is covered by a propeller domain, which excludes large peptides and proteins from the catalytic triad. Previous studies indicated that some amino acids of the N-terminal region constitute a part of the substrate entrance to the active site. To investigate the catalytic role of the N-terminus, we removed the residues 1-32 from the enzyme and examined the kinetic, thermodynamic, and structural consequences of the deletion, using the thermophile Pyrococcus furiosus prolyl oligopeptidase. An about threefold decrease in the catalytic activity along with a 20 degrees C reduction in the temperature optimum was observed. The pH-rate profile, the rate-limiting step, and the activation parameters did not change significantly. However, a substantial decrease was observed in the stability of the protein as demonstrated by circular dichroism and differential scanning calorimetry measurements, and by denaturation with guanidinium chloride. It was concluded that the N-terminal segment did not facilitate the substrate binding, independent of the size of the substrate, but contributed principally to the protein stability required for the formation of the proper active site.
Juhász
Tünde
T
Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest 112, Hungary.
Szeltner
Zoltán
Z
Polgár
László
L
D000818:Animals; D020134:Catalytic Domain; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D020027:Pyrococcus furiosus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D013552:Swine
D010446:Peptide Fragments; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases