The mechanism of adenosine deaminase catalysis studied by QM/MM calculations: The role of histidine 238 and the activity of the alanine 238 mutant

The initial rate determining step in the deamination of the natural substrate of adenosine deaminase is explored using hybrid quantum mechanical and molecular mechanical (QM/MM) methods. It is found that the intermediate formed following water activation by His 238 is energetically preferred over the one formed following activation by Asp 295. In the Ala 238 mutant a mechanism in which a second water molecule facilitates proton transfer gives rise to an intermediate whose stability relative to that for the wild type enzyme is close to that suggested from experimental kcat values. These results indicate that His 238 is the activating base and provide an explanation of the activity of the mutant. Alternative pathways for the reaction denoted electrophile- and substrate-activation are investigated and strong evidence is given favouring the latter, in which the intermediate is formed via nucleophilic attack of the protonated substrate.