MAGNESIUM ACTIVATION OF RIBONUCLEASE-H - EVIDENCE FOR ONE CATALYTIC METAL-ION

The metal ion dependence of Escherichia coli ribonuclease H activity h as been examined by monitoring the change in absorbance of nucleotide substrate by rapid (stopped-flow) kinetic methods. Kinetic equations t hat fully account for enzyme activation, and substrate inhibition at h igh metal concentration, have been derived. inhibition constants corre late with metal nucleotide binding affinities. Comparison of thermodyn amic (Huang, H.-W.; Cowan, J. A fur. J. Biochem. 1994, 219, 253-260) a nd kinetic data suggests that there is one essential catalytic metal c ofactor required for ribonuclease H activation, rather than a binuclea r magnesium site. This conclusion is in accord with recent crystallogr aphic data on the Mg2+-bound enzyme (Katayanagi, K.; Okumura, M.; Mori kawa, K. Proteins 1993, 17, 337-346). Similar conclusions are likely t o hold for the structurally homologous RNase H domains of retroviral r everse transcriptase.