Metallobiochemistry of the magnesium ion.

Ribonuclease H (Escherichia coli) contains one strong magnesium-binding site, as determined by metal-titration experiments monitored by high field ¹H-NMR and also by direct titration calorimetry. Kinetic and thermodynamic parameters were evaluated by ²⁵Mg-NMR and were as follows; dissociation constant K_d, ∼60±10 μM; activation free energy ΔG*, ∼49.8±0.9 kJ; on/off-rate for magnesium binding k_on, ∼ 1.8X1 ⁸ M ¹ s ¹, k_off, ∼ 1.1X1⁴ s ¹; quadrupole coupling constant Xa, 1.2±0.2 MHz. The dissociation constant was independently determined by standard analysis of ¹H chemical shifts in magnesium-titration experiments and by microcalorimetry (K_d∼20±20 μM). Cobalt hexamine, which also activates Rnase H [Jou, R. & Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685–6686], appears to bind at the same location as M ²⁺ (aqueous). Assignments of C2H and C4H protons to specific histidine residues have been made by two-dimensional correlated spectroscopy experiments. Direct ²⁵Mg-NMR pH titrations show that an ionizable residue (pK_a∼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 active-site chemistry are critically assessed and general physicochemical aspects of magnesium-binding sites on proteins and enzymes are discussed. [ABSTRACT FROM AUTHOR]