METALLOBIOCHEMISTRY OF THE MAGNESIUM-ION - CHARACTERIZATION OF THE ESSENTIAL METAL-BINDING SITE IN ESCHERICHIA-COLI RIBONUCLEASE-H

Ribonuclease H (Escherichia coli) contains one strong magnesium-binding site, as determined by metal-titration experiments monitored by high field H-1-NMR and also by direct titration calorimetry. Kinetic and thermodynamic parameters were evaluated by Mg-25-NMR and were as follows: dissociation constant K-d, approximate to 60 +/- 10 mu M; activation free energy Delta G*, approximate to 49.8 +/- 0.9 kJ; on/off-rate for magnesium binding k(on), approximate to 1.8 x 10(8) M(-1) s(-1), k(off), approximate to 1.1 x 10(4) s(-1); quadrupole coupling constant chi(B) 1.2 +/- 0.2 MHz. The dissociation constant was independently determined by standard analysis of H-1 chemical shifts in magnesium-titration experiments and by microcalorimetry (K-d approximate to 200 +/- 20 mu M). Cobalt hexaamine, which also activates RNase H [Jou, R. and Cowan, J. A. (1991) J. Am. Chem. Sec. 113, 6685-6686], appears to bind at the same location as Mg2+(aqueous). Assignments of C2H and C4H protons to specific histidine residues have been made by two-dimensional correlated spectroscopy experiments. Direct Mg-25-NMR pH titrations show that an ionizable residue (pK(a) approximate to 5.8), most likely one of the carboxylates in the active site, influences magnesium binding. On the basis of the magnesium coordination chemistry elucidated herein, recent proposals on activesite chemistry are critically assessed and general physicochemical aspects of magnesium-binding sites on proteins and enzymes are discussed.