Unusual Catalyst Concentration Effects in the Hydrolysis of Phenyl Phosphate Esters and of DNA: A Systematic Investigation of the Lanthanide Series¹

Abstract

Saturation kinetics are measured with all lanthanides and bis(nitrophenyl) phosphate BNPP as substrate. They show rather constant KM values; the kcat values, however, increase by up to 66 times for La3+ to Er3+ and decrease again for Yb3+ and Lu3+. With all lanthanides, hydrolysis of the intermediate mononitrophenyl phosphate NPP is 2−30 times faster than that of BNPP. The kcat values measured with BNPP correlate with the ion diameter of the lanthanides, in line with accepted mechanisms but with the notable exception of the higher lanthanides. A similar correlation holds for the cleavage rates with plasmid DNA, with striking differences again observed with the higher lanthanides, however. Thus, a concentration increase from 5 × 10-5 to 1 × 10-2 M leads to 64% and 84% more DNA cleavage with La3+ and Pr3+, respectively, but to up to 68% less DNA cleavage, respectively, but with Yb3+, Tm3+ or Lu3+. In contrast to the BNPP cleavage, saturation kinetics derived kcat values with DNA change little with the used cation, which on the other hand led to larger variations in the KM parameters. Preliminary UV and CD studies with plasmid DNA indicate lanthanide-induced conformational changes with pseudo-first-order rate constants 10−100 times higher than the cleavage rate under the same conditions. Again, Yb3+ shows different effects than Eu3+. The unusual behavior of the higher lanthanides is discussed on the basis of cation clustering, which, in contrast to earlier assumptions by Bamann et al., leads to diminished activities. Addition of salts such as of NaCl or MgCl2 leads to distinct decrease of catalytic effects of for instance Eu3+. The corresponding rates correlate well with Debye−Hückel ionic strength parameters. These as well as the effects of added amines are in line with a simple competition mechanism of the added cations for the anionic substrates.