Biological functions of the disulfides in bovine pancreatic deoxyribonuclease.
We characterized the biochemical functions of the small nonessential (C101-C104) and the large essential (C173-C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca(2+) protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca(2+), all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca(2+)-containing buffer. However, when diluted into a Ca(2+)-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65 degrees C, brDNase(C101A) at 60 degrees C, and brDNase(F192C/A217C) at 73 degrees C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca(2+)-containing buffer, 10%-18% of the bpDNase activity was restored, suggesting that the "essential" disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the "nonessential" disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (DeltaA650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction.
Chen
Wei-Jung
WJ
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei 10018, Taiwan. thliao@ccms.ntu.edu.tw
Lee
I-Shuan
IS
Chen
Ching-Ying
CY
Liao
Ta-Hsiu
TH
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002417:Cattle; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics*; D004220:Disulfides / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D007328:Insulin / Q000737:chemistry; D011489:Protein Denaturation; D040681:Structural Homology, Protein; D013879:Thioredoxins / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D004220:Disulfides; D007328:Insulin; D013879:Thioredoxins; D003850:Deoxyribonuclease I; D014357:Trypsin; D019791:Guanidine; D002118:Calcium