Kinetic and thermodynamic studies of purine represser binding to corepresser and operator DNA

Abstract

The kinetic and thermodynamic parameters for purine represser (PurR)- operator and PurR-guanine binding were determine using fluorescence spectroscopy and nitrocellulose filter binding. Operator binding affinity was increased by the presence of guanine as demonstrated previously (Choi, K. Y., Lu, F., and Zalkin, H. (1994) J. Biol. Chem. 269, 24066-24072; Rolfes, R. J., and Zalkin, H. (1990) J. Bacteriol. 172, 5637-5642), and conversely guanine binding affinity was increased by the presence of operator. Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding. Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepresser by ~10-fold. Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepresser ligand with a lower association rate constant than LacI binds to its inducer ligand. However, the rate constant for PurR-guanine binding to operator is ~3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulation by these two represser proteins provide a rationale for the observed functional differences.