Enzyme-catalyzed hydrolysis is a fundamental chemical transformation involved in many essential metabolic processes. The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of adenosine-containing metabolites in cysteine and methionine metabolism. Although MTAN enzymes contain highly similar active site architecture and generally follow a dissociative (DN*AN) reaction mechanism, substantial differences in reaction rates and chemical transition state structures have been reported. To understand how subtle changes in sequence and structure give rise to differences in chemistry between homologous enzymes, we have probed the reaction coordinates of two MTAN enzymes using quantum mechanical/molecular mechanical and molecular dynamics simulations combined with experimental methods. We show that the transition state structure and energy are significantly affected by the recruitment and positioning of the catalytic water molecule and that subtle differences in the noncatalytic active site residues alter the environment of the catalytic water, leading to changes in the reaction coordinate and observed reaction rate.