Mg²⁺-Free Bacillus stearothermophilus Tryptophanyl-tRNA Synthetase Retains a Major Fraction of the Overall Rate Enhancement for Tryptophan Activation

Abstract

Few experimental data are available for rates of enzymatic phosphoryl-transfer reactions in the absence of the divalent metal ions associated with such reactions. Such data are of interest for amino acid activation by class Ic aminoacyl-tRNA synthetases, for which there is substantial evidence that binding energy of ATP may account for a major fraction of the overall rate enhancement, and it is crucial to know if these effects themselves depend on the divalent metal ion. We describe a nested, non-linear model for the sum of metal-free and metal-catalyzed activities and its use in determining metal-free enzyme activity jointly with transition-state metal binding affinity, by fitting observed values obtained from Mg 2+ -depleted assays with increasing [EDTA] at known [Mg 2+ ] total . Tryptophan activation by B. stearothermophilus tryptophanyl-tRNA synthetase falls asymptotically to a plateau value five orders of magnitude below that observed for the Mg 2+ -supplemented enzyme at EDTA concentrations that reduce the free metal concentration to <1 pmolar. The fitted regression model parameters yield a relative rate acceleration of 9.3 × 10 4 attributable to the catalytic effect of Mg 2+ and an enhanced (K E ‡ = 1.15 × 10 −7 M) transition-state binding of Mg 2+ . Factorial analysis indicates that 80% of the reduction in free energy of activation effected by TrpRS arises from proteinligand interactions.

Combining multi-mutant and modular thermodynamic cycles to measure energetic coupling networks in enzyme catalysis

“…The stunning quantitative agreement between the coupling energies estimated from multi-mutant and modular combinatorial experiments with those anticipated from the characterization of metal-free catalysis 25 reinforces our earlier suggestions 32,34,38,66 that domain motions are necessary to form the TrpRS·transition state complex, and hence that differences observed between TrpRS crystal structures are relevant to the catalytic cycle. Thus, the different TrpRS crystal structures probably approximate those states connected by motions necessary for transition-state stabilization.…”

“…Prompted by the suggestion that the TrpRS catalysis should be essentially eliminated in the absence of Mg 2+ ion, we prepared extensively depleted stocks of enzyme, assay buffer, and reagents 25 . The resulting assays exhibited reduced, but still substantial rate accelerations over the uncatalyzed rate, which we initially attributed to residual remaining Mg 2+ ion (Fig.…”

“…Iterated solution of the simultaneous binding equilibria using known dissociation constants for the Mg 2+ ·ATP and Mg 2+ ·EDTA equilibria allowed us to fit the activity data at two TrpRS concentrations to a nonlinear model with two parameters: the proportional decrease in rate in the absence, relative to that in the presence of Mg 2+ ion (1.1 × 10 −5 ) and the dissociation constant of Mg 2+ from the transition-state complex (120 nM) 25 (Fig. 3(c)).…”

“…Testing that idea experimentally, we found that only about a third of the overall transition-state stabilization could be attributed to the Mg 2+ ion 25 . This posed two questions:

What accounts for the metal-independent two-thirds of the transition-state stabilization?

How does metal-protein coupling work if Mg 2+ does not interact with the protein, has very little effect on the rate in water, but accelerates the rate 10 5 -fold in the presence of TrpRS?

…”

Coding of Class I and II Aminoacyl-tRNA Synthetases

“…Mechanistic studies on intact TrpRS had previously identified a profound intramolecular coupling, ΔΔG ‡ ~ − 5 kcal/mole necessary for catalytic assist by the active-site Mg 2+ ion [92] and achieving full catalytic proficiency by the full-length enzyme [89,32,93,92]. A five-way coupling interaction, ΔΔG ‡ ~ − 5kcal/mole, was also measured between four residues in an allosteric switching region 20 Å from the active-site metal that mediates the shear involved in domain movement during catalysis [94].…”