Mechanistic Enzymology

 
 

The Class I TrpRS couples large-scale domain movement tightly to catalysis. Amino acid side chains involved in the shear associated with domain movement (yellow) communicate their status to the active-site Mg2+ ion (white; 20 Å away), thereby determining whether or not it contributes to catalysis. This Mg2+-assisted catalysis supplements distinct transition-state stabilization mechanisms that involve the conserved peptide signatures HIGH (behind ATP) and KMSKS (red).


Documenting this mechanism involved measuring the energetic contribution of the metal to the overall rate acceleration, showing that this rate acceleration requires protein-metal coupling, identifying and mutating the residues whose neighbors change during domain movement, and then experimentally measuring the differential conformational stabilization of intermediate conformational states during catalysis together with a full factorial analysis of higher-order coupling between these sites and the catalytic metal ion by assaying all 15 combinatorial mutants with both Mg2+ and Mn2+. Analysis of the coupling revealed that virtually all of the catalytic effect of Mg2+ arises from the five-way interaction with the remote site, which we call the D1 switch. See more details.

Players and Process

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