The Impact of Perturbing Dynamic Amino Acid Networks in a (β/α)8 Barrel Enzyme

Abstract

Amino acid networks describe the web of noncovalent interactions between residues spanning an enzyme. These networks may be responsible for the propagation of regulatory signals across the protein that influence conformation, binding of substrate(s), and catalysis. Tryptophan synthase (TS), the final enzyme in the tryptophan biosynthetic pathway, is a tetramer consisting of a pair of alpha and beta heterodimers arranged in a linear conformation. The alpha and beta subunits are connected by a 25Å intramolecular tunnel that channels indole, a product from the alpha reaction, to the active site in the beta subunit. In addition to this tunnel, the conformational states of these subunits are highly coordinated making TS an ideal and heavily studied model for substrate channeling and enzyme-enzyme interactions. We used nuclear magnetic resonance chemical shift covariance analysis to delineate amino acid networks in the alpha subunit, a (β/α)8 barrel enzyme. We have shown that these observed networks change between the resting state (in the absence of substrates) and the working state (under active catalytic turnover). Furthermore, the loss of a hydrogen bond between the dynamic β2α2 and β6α6 loops in the T183V variant significantly changes these networks and the catalytic rate seen in the wild-type. These networks were perturbed by making small Ala to Gly substitutions of surface residues correlated to the catalytic Glu49. These modifications resulted in modest decreases in the catalytic rate, although they are 25 Å away from the active site. Amino acid networks are important for the function of an enzyme and may be manipulated to tune its function.