KINETIC AND THERMODYNAMIC STUDIES OF PURINE REPRESSOR BINDING TO COREPRESSOR AND OPERATOR DNA

The kinetic and thermodynamic parameters for purine repressor (PurR)-o perator and PurR-guanine binding were determined using fluorescence sp ectroscopy and nitrocellulose filter binding. Operator binding affinit y 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, 5637-56 42), and conversely guanine binding affinity was increased by the pres ence of operator, Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate consta nt for binding. Operator had minimal effect on the association rate co nstant for guanine binding; however, this DNA decreased the dissociati on rate constant for corepressor by similar to 10-fold, Despite signif icant sequence and structural similarity between PurR and LacI protein s, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand, However, the rate cons tant for PurR-guanine binding to operator is similar to 3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulati on by these two repressor proteins provide a rationale for the observe d functional differences.