The catalytic effects of perdeuterating the pyridoxal phosphate-dependent enzyme alanine racemase from Geobacillus stearothermophilus are reported. The mass of the heavy perdeuterated form is similar to 5.5% greater than that of the protiated form, causing kinetic isotope effects (KIEs) of similar to 1.3 on k(cat) and k(cat)/K-M for both L- and D-alanine. These values increase when C alpha-deuterated alanine is used as the substrate. The heavy-enzyme KIEs of similar to 3 on k(cat)/K-M with deuterated substrates are greater than the product of the individual heavy-enzyme and primary substrate KlEs. This breakdown of the rule of the geometric mean is likely due to coupled motion between the protein and the proton-transfer reaction coordinate in the rate-limiting step. These data implicate a direct role for protein vibrational motions in barrier crossing for proton-transfer steps in alanine racemase.