Identification of three crucial histidine residues (His115, His132 and His297) in porcine deoxyribonuclease II.
DNase II is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its catalytic mechanism, we constructed plasmids encoding nine different histidine (H)-to-leucine (L) mutants for porcine DNase II and examined the enzyme properties of the expressed mutant proteins. Of the mutants, all but H132L were secreted into the medium of expressing cells. Six of the mutated DNase II proteins (H41L, H109L, H206L, H207L, H274L and H322L) showed enzyme activity, whereas the H115L, H132L and H297L mutants exhibited very little activity. The H115L and H297L mutants were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II mutants; these mutants were inactive, but their DNase activities could be rescued with imidazole, indicating that His115 and His297 are likely to function as a general acid and a general base respectively in the catalytic centre of the enzyme. In contrast with the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for His132 with lysine resulted in the misfolded form being retained in endoplasmic reticulum.
Cheng
Yu-Che
YC
Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Hsueh
Chin-Chen
CC
Lu
Shao-Chun
SC
Liao
Ta-Hsiu
TH
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
D011994:Recombinant Proteins; D006639:Histidine; D004247:DNA; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II