Ca2+-dependent activity of human DNase I and its hyperactive variants.
We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double-stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300-fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild-type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+-dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild-type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26-fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate-enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.
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Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA.
Lazarus
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D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation
D004278:DNA, Superhelical; D007527:Isoenzymes; D004247:DNA; D003850:Deoxyribonuclease I; D002118:Calcium