Thermodynamic analysis of inducer binding to the lactose repressor protein: contributions of galactosyl hydroxyl groups and beta substituents

Abstract

Kinetic and equilibrium studies of the binding of modified beta-D-galactoside sugars to the lac repressor were carried out to generate thermodynamic data for protein-inducer interactions. The energetic contributions of the galactosyl hydroxyl groups to binding were assessed by using a series of methyl deoxyfluoro-beta-D-galactosides. The C-3 and C-6 hydroxyls contributed less than or equal to -2.3 and -1.7 +/- 0.3 kcal/mol to the binding free energy change, respectively, whereas the C-4 hydroxyl provided only a nominal contribution (-0.1 +/- 0.2 kcal/mol). Favorable contributions to the total binding free energy change were observed for replacement of O-methyl by S-methyl at the beta-anomeric position and for S-methyl by S-isopropyl. Negative delta H degrees values characteristic of protein-sugar complexes [Quiocho, F. A. (1986) Annu. Rev. Biochem. 55, 287-315] were observed for a series of beta-D-galactosides differing at the beta-glycosidic position. A decrease in delta H degrees of approximately 6 kcal/mol upon replacement of the O-methyl substituent by S-methyl indicates a substantial increase in van der Waals' interactions and/or hydrogen bonding in this region of the ligand binding site. The more favorable free energy change for the binding of the S-isopropyl vs S-methyl compound is due mainly to more positive entropic contributions, consistent with an increase in apolar interactions.(ABSTRACT TRUNCATED AT 250 WORDS)