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-bindin g site, as determined by metal-titration experiments monitored by high field H-1-NMR and also by direct titration calorimetry. Kinetic and t hermodynamic parameters were evaluated by Mg-25-NMR and were as follow s: dissociation constant K-d, approximate to 60 +/- 10 mu M; activatio n free energy Delta G, approximate to 49.8 +/- 0.9 kJ; on/off-rate fo r magnesium binding k(on), approximate to 1.8 x 10(8) M(-1) s(-1), k(o ff), approximate to 1.1 x 10(4) s(-1); quadrupole coupling constant ch i(B) 1.2 +/- 0.2 MHz. The dissociation constant was independently dete rmined by standard analysis of H-1 chemical shifts in magnesium-titrat ion experiments and by microcalorimetry (K-d approximate to 200 +/- 20 mu M). Cobalt hexaamine, which also activates RNase H [Jou, R. and Co wan, J. A. (1991) J. Am. Chem. Sec. 113, 6685-6686], appears to bind a t the same location as Mg2+(aqueous). Assignments of C2H and C4H proto ns to specific histidine residues have been made by two-dimensional co rrelated 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 herei n, recent proposals on activesite chemistry are critically assessed an d general physicochemical aspects of magnesium-binding sites on protei ns and enzymes are discussed.