Highly Efficient Cofactors of Cu²⁺-Dependent Deoxyribozymes

Deoxyribozymes can catalyze various types of reactions mostly with metal ions as their cofactors. Here we demonstrate that three copper compounds with thiosemicarbazide or semicarbazide ligands, are highly active cofactors of Cu²⁺-dependent deoxyribozymes without any additional reagents. The maximum catalytic rate constant of the deoxyribozyme with dichloro(di-thiosemicarbazide)copper as a cofactor is ∼25-fold faster than Cu²⁺ cofactor under the same reaction condition. Using a variety of spectroscopies, electrochemistry, and mutagenesis, we demonstrate that both the three-dimensional structure and redox potential of the copper center of the cofactors are essential to the catalysis of deoxyribozymes. The cofactor interacts with the enzyme-substrate complex forming a ternary enzyme-substrate-cofactor complex, which is confirmed by changing the structure of the enzyme-substrate complex through varying the temperature, or mutating the active site of the enzyme and conserved site of the substrate. The result suggests that Cu²⁺-dependent deoxyribozyme requires a well-defined active site to carry out the catalysis proficiently.