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Lanthanide ions as required cofactors for DNA catalysts.

ABSTRACT: We report that micromolar concentrations of lanthanide ions can be required cofactors for DNA-hydrolyzing deoxyribozymes. Previous work identified deoxyribozymes that simultaneously require both Zn(2+) and Mn(2+) to achieve DNA-catalyzed DNA hydrolysis (10(12) rate enhancement); a mutant of one such DNA catalyst requires only Zn(2+). Here we show that in vitro selection in the presence of 10 µM lanthanide ion (Ce(3+), Eu(3+), or Yb(3+)) along with 1 mM Zn(2+) leads to numerous DNA-hydrolyzing deoxyribozymes that strictly require the lanthanide ion as well as Zn(2+) for catalytic activity. These DNA catalysts have a range of lanthanide dependences, including some deoxyribozymes that strongly favor one particular lanthanide ion (e.g., Ce(3+) >> Eu(3+) >> Yb(3+)) and others that function well with more than one lanthanide ion. Intriguingly, two of the Yb(3+)-dependent deoxyribozymes function well with Yb(3+) alone (K(d,app) ~10 µM, in the absence of Zn(2+)) and have little or no activity with Eu(3+) or Ce(3+). In contrast to these selection outcomes when lanthanide ions were present, new selections with Zn(2+) or Mn(2+) alone, or Zn(2+) with Mg(2+)/Ca(2+), led primarily to deoxyribozymes that cleave DNA by deglycosylation and ?-elimination rather than by hydrolysis, including several instances of depyrimidination. We conclude that lanthanide ions warrant closer attention as cofactors when identifying new nucleic acid catalysts, especially for applications in which high concentrations of polyvalent metal ion cofactors are undesirable.

SUBMITTER: Dokukin V

PROVIDER: S-EPMC3521562 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Lanthanide ions as required cofactors for DNA catalysts.

Dokukin Victor V Silverman Scott K SK

Chemical science 20120301 5

We report that micromolar concentrations of lanthanide ions can be required cofactors for DNA-hydrolyzing deoxyribozymes. Previous work identified deoxyribozymes that simultaneously require both Zn(2+) and Mn(2+) to achieve DNA-catalyzed DNA hydrolysis (10(12) rate enhancement); a mutant of one such DNA catalyst requires only Zn(2+). Here we show that in vitro selection in the presence of 10 µM lanthanide ion (Ce(3+), Eu(3+), or Yb(3+)) along with 1 mM Zn(2+) leads to numerous DNA-hydrolyzing de ...[more]

PMID: 23243490

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Project description:Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm(3+), whose ionic radius is closest to that of Ca(2+), is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100mum in the presence of 5mm-Ca(2+). Er(3+) and Lu(3+), which both have ionic radii smaller than either Ca(2+) or Sm(3+), inhibit less efficiently, and La(3+), which is slightly larger than Ca(2+) or Sm(3+), inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca(2+)-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca(2+) [ionic radius 0.099nm (0.99 A)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca(2+), requires lanthanide concentrations 50-100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50mum, Sm(3+) lowers the apparent K(m) for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent V(max.) from 0.29 Wünsch-Heidrich unit to 0.018 unit. Thus Sm(3+) enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme-Sm(3+)-substrate complex. Inhibition by Sm(3+) is competitive with regard to Ca(2+). The apparent dissociation constant, K(d), of Ca(2+) is 0.27mm, where the K(i) for Sm(3+) is 12mum. Clostridiopeptidase is more thermolabile in the absence of Ca(2+). With Sm(3+), thermoinactivation of the enzyme at 53 degrees C or 60 degrees C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca(2+) at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.

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Lanthanide ions as required cofactors for DNA catalysts.

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Lanthanide ions as required cofactors for DNA catalysts.

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