Synthesis of [3'-H-2)-labeled 5'-methylthioadenosine (MTA) derivatives permitted measurement of the [3'-H-2) KIE of the reaction catalyzed by Streptococcus pneumoniae methylthioadenosine nucleosidase (spMTAN). The key [3'-H-2) KIE revealed the partial 3'-OH polarization and H3'-endo -> exo ribosyl configuration at the spMTAN transition state. A new understanding of the transition state stabilization of spMTAN-catalyzed hydrolysis is uncovered in structural features at the spMTAN transition state.