INITIAL-STATE OF AN ENZYMATIC-REACTION - THEORETICAL PREDICTION OF COMPLEX-FORMATION IN THE ACTIVE-SITE OF RNASE T1

A computer model for the hydrated complex between the enzyme ribonuclease (RNase) T1 and its substrate guanylyl-3',5'-guanosine has been refined using molecular dynamics simulation and quantum chemical calculations. Actual protonation states of the most important residues at the active site in the presence of the substrate were derived from published NMR titrations and pH-dependent kinetic studies, which were confirmed by independent Monte Carlo calculations (manuscript in preparation). The molecular dynamics trajectory has been analyzed to theoretically capture the initial point of the enzymatic reaction pathway. The changes in the charge distribution of the most relevant part of the enzyme-substrate complex have been checked by the CNDO/2-spd technique. The initial point of the enzymatic reaction pathway has been found to correspond as expected, to the strained conformation of the ''substrate + active site side chains'' complex. His40, Glu58, Arg77, and His92 which are primarily involved in the enzymatic activity show hydrogen bond contacts to the substrate. In this scheme, Glu58 plays the role of general base and His92 acts as the general acid in the reaction pathway, while the other two residues stabilize the initial state of the reaction electrostatically.