Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Reversible binding of a ligand to an enzyme active site can elicit a variety of changes in the protein, such as conformational changes (close to the site of binding or communicated over long distances), changes in the ionization state of surrounding amino acid side chains, changes in the interaction of the target protein with other subunits (or other proteins), or even changes in the thermodynamic stability of the protein. Relatively little attention has been given to studying these effects in proteins to which the ligand has been irreversibly bound, yet this can be a convenient way of studying the effects of ligand binding in the absence of association/dissociation equilibria. We report the dramatic changes which occur to the shikimate pathway enzyme dehydroquinase when ligand is attached to its active site after borohydride reduction of the mechanistically important Schiff's base intermediates. The effects of this modification have been characterized by limited proteolysis, circular dichroism, guanidine hydrochloride denaturation, and differential scanning calorimetry. The conclusions from these studies are that although anchoring the ligand at the active site does not cause a gross change in conformation, it does increase markedly the conformational stability of the protein. This is conclusively established by three separate experiments: 1) the modified protein is completely resistant to proteases, whereas the unmodified protein is very susceptible to proteolysis; 2) the concentration of guanidine hydrochloride required to unfold the ligand-linked dehydroquinase is 3-4-fold greater than that of the unmodified protein; 3) the melting temperature (Tm) of the modified protein is 40 degrees C higher than that of the unmodified protein. These results are a very clear example of the thermodynamic link between ligand binding, conformational stability, and proteolytic susceptibility in vitro and will be a useful system for dissecting the contributions of individual protein-ligand interactions to these parameters.