Probing the general base catalysis in the first step of BamHI action by computer simulations

BamHI is a type II restriction endonuclease that catalyzes the scission of the phoshodiester bond in the GAGTCC cognate sequence in the presence of tw o divalent metal ions. The first step of the reaction is the preparation of water for nucleophilic attack by Glu-113, which has been proposed to abstr act the proton from the attacking water molecule. Alternatively, the 3'-pho sphate group to the susceptible phosphodiester bond has been suggested to p lay a role as the general base. The two hypotheses have been tested by comp uter simulations using the semiempirical protein dipoles Langevin dipoles ( PDLD/S) method. Deprotonation of water by Glu-113 has been found to be less favorable by 5.7 kcal/mol than metal-catalyzed deprotonation with a concom itant proton transfer to bulk solvent. The preparation of the nucleophile b y the 3'-phosphate group is less favorable by 12.3 kcal/mol. These results suggest that both the general base and the substrate-assisted mechanisms in the first step of BamHI action are less likely than the metal-catalyzed re action. The metal ions in the active site of BamHI make the largest contrib utions to the reduction of the free energy of hydroxide ion formation. On t he basis of these findings we propose that the first step of endonuclease c atalysis does not require a general base; rather, the essential attacking n ucleophile in BamHI catalytic action is stabilized by the metal ions.