Heavy Enzyme Kinetic Isotope Effects on Proton Transfer in Alanine Racemase

摘要

The catalytic effects of perdeuterating the pyridoxal phosphate dependent enzyme alanine racemase from Geobacillus stearothermophilus are reported. The heavy, perdeuterated form is ~5.5% greater in mass than the protiated form, causing kinetic isotope effects (KIEs) of ~1.3 on kcat and kcat/KM for both L- and D-alanine. These values increase when Cα-deuterated alanine is used as substrate. The heavy enzyme KIEs of ~3 on kcat/KM 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.