Metal-ion-promoted dephosphorylation of the 5'-triphosphates of uridine and thymidine, and a comparison with the reactivity in the corresponding cytidine and adenosine nucleotide systems

Abstract

First-order rate constants (50 degrees C; I = 0.1 M, NaClO4) for the dephosphorylation of UTP and TTP (1 mM) in the pH range 2-10 are compared with those of ATP and CTP; they all show the same properties indicating that the nucleic base has no influence on the rate. In the presence of Cu2+ or Zn2+ (NTP:M2+ = 1:1) this changes drastically: ATP-M2+ much greater than UTP-M2+ approximately equal to TTP-M2+ approximately equal to CTP-M2+ greater than NTP, the Cu2+ systems being always more reactive than the Zn2+ systems, and these more than the Ni2+ systems. An interaction between the nucleic base and metal ion is important for the Cu2+-ATP and Zn2+-ATP systems, but not for the pyrimidine-nucleotide systems (these behave like methyltriphosphate). Accordingly, prevention of the Cu2+-purine interaction by the addition of one equivalent of 2,2'-bipyridyl, leading to Cu(Bpy) (NTP)2-, strongly reduces the activity and all four ternary Cu2+ systems now show the same dephosphorylation rate. Addition of a second equivalent of Cu2+ to the Cu2+-UTP 1:1 system enhances the dephosphorylation rate significantly and Job's method provides evidence that a 2:1 complex is the most reactive intermediate. The relation between the initial rate, vo = d[PO3-4]/dt, and the concentration of Cu2+-UTP in 1:1 and 2:1 systems was determined. The results suggest that the reactive complex with pyrimidine nucleotides is a monomeric, dinuclear species of the type M2(NTP) (OH)- (its formation is inhibited by ligands like tryptophanate), while with M2+-ATP the reactive complex is a dimer. The connection between the indicated dephosphorylations in vitro, i.e. trans-phosphorylations to H2O, and related reactions in vivo are discussed.