title: Thermodynamic analysis of the equilibrium, association and dissociation of 2'GMP and 3'GMP with ribonuclease T1 at pH 5.3.
abstract: Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.
authors.affiliation: Department of Medical Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: A D
authors.initials: AD
authors.lastname: MacKerell
authors.forename: R
authors.initials: R
authors.lastname: Rigler
authors.forename: U
authors.initials: U
authors.lastname: Hahn
authors.forename: W
authors.initials: W
authors.lastname: Saenger
mesh_terms: D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004058:Diffusion; D006157:Guanosine Monophosphate / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D006163:Ribonuclease T1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
chemical_list: C026693:2'-guanylic acid; C068152:3'-guanylic acid; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1