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 nonequilibrium molecular dynamics simulations, multivariant 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 a-phosphate. However, net coupling to the metal opposes catalysis in both ground (KM) and transition (k(cat)) states. The 10(5)-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 F371 mutation, which both reduces k(cat) 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.