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 .ltoreq. 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.degree. values characterized of protein-sugar complexes [Quiocho, F. A. (1986) Ahnu. 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.degree. of .apprx. 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. Thermodynamic parameters for isopropyl .beta.-D-thiogalactoside (IPTG) binding at neutral pH are in agreement with previously published results [Butler, A. P., Revzin, A., and von Hippel, P. H. (1977) Biochemistry 16, 4757-4758; Donner, J., Caruthers, M. H., and Gill, S. J. (1982) J. Biol. Chem. 257, 14826-14829]. Arrhenius plots of kinetic rate constants for the binding of IPTG, methyl .beta.-D-galactoside, and methyl .beta.-D-thiogalactoside to the repressor revealed a protein structural transition at 12.degree. C. All of the experimental data are consistent with the hypothetical sugar binding site for repressor protein proposed by Sams et al. (1984) [Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature (London) 310, 429-430].