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.
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.
Elofsson
A
A
Department of Medical Biophysics, Karolinska Institute, Stockholm, Sweden.
Kulinski
T
T
Rigler
R
R
Nilsson
L
L
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
D015226:Dinucleoside Phosphates; D008024:Ligands; D015075:2-Aminopurine; C068152:3'-guanylic acid; D006157:Guanosine Monophosphate; D006163:Ribonuclease T1