Conditional Mg 2+-Assisted Catalysis: A Master Switching Motif Responsible for Differential Stability Suggests a General Transducing Mechanism

Abstract

B. stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) uses different conformational states to catalyze tryptophan activation. A single Mg2+ ion increases transition state stabilization by −6.5 kcal/mol for optimal catalysis. Catalytic assist occurs if, and only if, the Mg2+ interacts with the protein. We are trying to identify the metal-protein interactions that produce this catalytic effect. Physical interactions between Mg2+ and TrpRS are mediated indirectly via active-site lysines K111, K192 and K195. Mutations of these lysines showed that they all stabilize the transition state. However, their interactions with the Mg2+ significantly reduce their catalytic effects. Catalytically productive interactions between TrpRS and the Mg2+ ion must therefore arise from outside the active site. We identified a core set of residues we call the D1 switch because they move during the catalytic conformational transition. The D1 switch lies at the corner of the N-terminal ß-α-ß crossover distal to the active site. It is highly conserved in Rossmannoid proteins. The Rosetta Design program suggested that mutations of D1 residues could ‘‘hyperstabilize’’ the activated state observed just prior to catalysis. Multimutant thermodynamic cycles together with substitution of Mn2+ for Mg2+ and [ATP]-dependent Michaelis-Menten kinetics demonstrate significant long-range synergistic coupling between the D1 switch and the Mg2+ ion. Thus, long-range interactions to the metal likely drive catalysis indirectly, by changing an inactive Mg2+ coordination into one that can stabilize the transition state. In this way transition-state stabilization by Mg2+ occurs if, and only if, conformational changes reposition it. We suggest that other NTPase enzymes may use similar conditional activation of Mg2+ to couple catalyzed hydrolysis of their purine triphosphate substrates to conformational changes, thereby transducing chemical free energy for cellular work and signaling. Supported by NIGMS 78227, 90406.