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°C, brDNase(C101A) at 60°C, and brDNase(F192C/A217C) at 73°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 (⌬A650nm/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.

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The two amino acid residues, Asp 99 and Asp 201, involved in the coordination of the two calcium atoms in the X-ray structure of bovine pancreatic (bp) DNase, were individually changed by site-directed mutagenesis. The two altered proteins, brDNase(D99A) and brDNase(D201A) were expressed in Escherichia coli and purified by anion exchange chromatography. Equilibrium dialysis showed that mutation destroyed one Ca 2+ -binding site each in brDNase(D99A) and brDNase(D201A). Compared with bpDNase, the Vmax value for brDNase(D99A) remained unchanged and that for brDNase(D201A) was decreased, whereas the K m values for the two variants were increased two-to threefold when the DNA hydrolytic hyperchromicity assay was used. Like bpDNase, brDNase(D99A) was able to make double scission on duplex DNA with Mg 2+ plus Ca 2+ and was effectively protected by Ca 2+ from the trypsin inactivation. But under the same conditions, brDNase(D201A) lost the double-scission ability and was not protected by Ca 2+ . Nevertheless, the two variant proteins retained the characteristics of the Ca 2+ -induced conformational changes and the Ca 2+ protection against the ␤-mercaptoethanol disruption of the essential disulfide bond, suggesting that other weaker Ca 2+ -binding sites not found in the X-ray structure were responsible for these properties. Therefore, the two structural calcium atoms are not for maintaining the overall conformation of the active DNase, as it has been indicated in the X-ray analysis, but rather play the role in the fine-tuning of the DNase activity.