Ab initio molecular orbital theory was used to examine the role of active site tyrosine in glutathione S-transferases by using appropriate model systems. The location of the key mechanistic proton of the enzyme-glutathione binary complex, O- - -H- - -S, was predicted to be near the phenolic oxygen, which is in agreement with experiments. However, the position of the proton can be manipulated by changing the acidity of the tyrosine, which can be accomplished by either introducing a substituent group to the tyrosine phenol ring or changing the protein environment. Thus, our study seems to have resolved previous confusion as to where the proton is located. The hydrogen bonding between tyrosine and thiolate of glutathione is very strong. On the basis of our present study, we propose that, in the Y6F (Tyr --> Phe) mutant, a water molecule replaces the function of the hydroxyl group of the active site tyrosine of the wild-type enzyme. Several lines of evidence in support of the above hypothesis are discussed. The latter hypothesis is reminiscent of the notion of substrate-assisted catalysis.