SITE-SPECIFIC POINT MUTATION CHANGES SPECIFICITY - A MOLECULAR MODELING STUDY BY FREE-ENERGY SIMULATIONS AND ENZYME-KINETICS OF THE THERMODYNAMICS IN RIBONUCLEASE T(1) SUBSTRATE INTERACTIONS

We have theoretically and experimentally studied the binding of two di fferent ligands to wild-type ribonuclease T1 (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln. The binding of the natural substrat e 3'-GMP has been compared with the binding of a fluorescent probe, 2- aminopurine 3'-monophosphate (2AP), and relative free energies of bind ing of these ligands to the mutant and the wild-type (wt) enzyme have been calculated by free energy perturbation methods. The free energy p erturbations predict that the mutant RNT1-Gln-46 binds 2AP better than 3'GMP, in agreement with experiments on dinucleotides. Four free ener gy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Becaus e of the larger number of atoms involved, it was necessary to use a mu ch longer simulation time for the change in the protein, i.e., the per turbation from Glu to Gln, than in the perturbation from 3'-GMP to 2AP . Finally the structure of the binding site is analyzed for understand ing differences in catalytic speed and binding strength. (C) 1993 Wile y-Liss, Inc.