Abstract
Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this working state represents a four to one ratio of the enzyme bound with the indole-3-glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde-3-phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate- and products-bound states to the working state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in the working state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in the working state. As the enzyme transitions from the substrate-bound to the products-bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit.