Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase

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 ∼5.5% greater than that of the protiated form, causing kinetic isotope effects (KIEs) of ∼1.3 on k _cat and k_cat/K_M for both l-and d-alanine. These values increase when Cα-deuterated alanine is used as the substrate. The heavy-enzyme KIEs of ∼3 on k_cat/K_M with deuterated substrates are greater than the product of the individual heavy-enzyme and primary substrate KIEs. 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.