A new method had previously been developed for the testing of the primary and secondary hydrogen coupling along a reaction coordinate and dihydrofolate reductase (DHFR) has been used as the model system. Secondary kinetic isotope effects have been competitively measured for the double mutant (G121V-M42W) DHFR, with protium and deuterium transfer from primary position. The 2ºKIE values were measured when the isotopically labeled atom was not involved in the bond cleavage step and served as sensitive probes of the nature of the chemical transformation. The results have shown that isotopic substitution at the primary position had no effect on the hybridization change at the secondary position. Such findings were in agreement with the rule of geometric mean, providing no evidence of 1º-2º coupled motion. Similar experiments on the wild-type and two single mutants (G121V and M42W DHFR) have also indicated no coupling. The results showed a possible different physical nature of the H-transfer step compared to the single mutants. The investigation of G121V-M42W DHFR have addressed the long term standing hypothesis that these two residues are part of the dynamic network that is related to the catalyzed H-transfer