Mg2+‐Assisted Catalysis by B. stearothermophilus TrpRS is Promoted by Allosteric Effects

Abstract

Mg2+ ion accelerates TrpRS‐catalyzed Trp activation ~105 fold, providing 35% of the reduction in ΔG‡. This catalytic contribution is achieved indirectly, by interactions from active‐site lysine residues to the non‐bridging phosphate oxygen atoms that coordinate the Mg2+, lengthening metal‐oxygen distances and weakening interaction with the triphosphate. This electrostatic compromise is coupled to an endergonic twist of the anticodon‐binding domain relative to the active site, whose unfavorable energetics arise from long‐range interactions, and which leads to (exergonic) untwisting during the catalytic step. We investigated these interactions quantitatively, determining mutant effects in multi‐variant thermodynamic cycles with [ATP]‐dependent Michaelis‐Menten kinetics and Mn2+ for Mg2+ substitution. These cycles confirm the coupling of lysines to metal coordination and rule out a proposed interaction between the metal and the nearly invariant aspartate, D146. The two lysine residues most strongly coupled to the metal bind the PPi leaving group; the third interacts with the Pα phosphate. Coupling was also observed for a remote mutation, F37I, which affects both kcat and the use of Mn2+. Tηεσειντεραχτιℴνσ αρε χℴνσιστεντ ωιτη στραιν αρισινγ φρℴμ αν υνφαϖℴραβλε χℴνφℴρματιℴν νεχεσσαρψ τℴ ασσεμβλε τηε αχτιϖε σιτε αρℴυνδδ A TΠ ανδ ωηιχη εσταβλισηεσ α δελιχατε βαλανχε ℴφ ελεχτρℴστατιχ ανδ μεχηανιχαλ φℴρχεσ τηατ στραιν τηε σχισσιλε Πα‐O‐Pβ linkage. Supported by NIGMS 48519 and 45295.