Increased proteolytic resistance of ribonuclease A by protein engineering.
Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.
Markert
Y
Y
Department of Biochemistry/Biotechnology, Martin-Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.
Köditz
J
J
Mansfeld
J
J
Arnold
U
U
Ulbrich-Hofmann
R
R
D000409:Alanine / Q000737:chemistry; D000818:Animals; D002942:Circular Dichroism; D019286:Endopeptidase K / Q000378:metabolism*; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology; D010196:Pancreatic Elastase / Q000378:metabolism*; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012394:Rosaniline Dyes; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000378:metabolism*; D013552:Swine; D013816:Thermodynamics
D012394:Rosaniline Dyes; D011392:Proline; D012259:Ribonuclease, Pancreatic; D013381:Subtilisins; D010196:Pancreatic Elastase; D019286:Endopeptidase K; C004692:coomassie Brilliant Blue; D000409:Alanine