title: Site specific point mutation changes specificity: a molecular modeling study by free energy simulations and enzyme kinetics of the thermodynamics in ribonuclease T1 substrate interactions.
abstract: We have theoretically and experimentally studied the binding of two different ligands to wild-type ribonuclease T1 (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln. The binding of the natural substrate 3'-GMP has been compared with the binding of a fluorescent probe, 2-aminopurine 3'-monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild-type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1-Gln-46 binds 2AP better than 3'GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i,e., the perturbation from Glu to Gln, than in the perturbation from 3'-GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength.
authors.affiliation: Department of Medical Biophysics, Karolinska Institute, Stockholm, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Elofsson
authors.forename: T
authors.initials: T
authors.lastname: Kulinski
authors.forename: R
authors.initials: R
authors.lastname: Rigler
authors.forename: L
authors.initials: L
authors.lastname: Nilsson
mesh_terms: D015075:2-Aminopurine / Q000378:metabolism; D003198:Computer Simulation; D015226:Dinucleoside Phosphates / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D017354:Point Mutation; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D015226:Dinucleoside Phosphates; D008024:Ligands; D015075:2-Aminopurine; C068152:3'-guanylic acid; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1