Mg2+-Assisted Catalysis by B. Stearothermophilus TrpRS Is Promoted by Allosteric Effects

Mn2+-assisted catalysis by B. stearothermophilus TrpRS parallels that in polymerases and reduces specificity in amino acid activation. As predicted by non-equilibrium molecular dynamics simulations, multi-variant thermodynamic cycles with [ATP]-dependent Michaelis-Menten kinetics and Mn2+ for Mg2+ substitution demonstrate energetic coupling of ATP affinities to the metal; to lysines K111 and K192, which interact via the PPi leaving group; and to K195, which couples differently to the metal via the α–phosphate. However, net coupling to the metal opposes catalysis in both ground (Km) and transition (kcat) states. The 105-fold rate acceleration by Mg2+-protein interactions therefore requires additional favorable protein-metal couplings. Examples include longer-range, i.e., allosteric, interactions previously illustrated by the remote F37I mutation, which both reduces kcat and enhances catalytic assist by Mn2+, relative to that by Mg2+. These data support a model linking metal-assisted phosphoryl transfer catalysis to domain movement, and hence to free-energy transduction in a broad range of enzymes