Abstract
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 kcat/KM 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 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.
Original language | English (US) |
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Pages (from-to) | 2509-2511 |
Number of pages | 3 |
Journal | Journal of the American Chemical Society |
Volume | 135 |
Issue number | 7 |
DOIs | |
State | Published - Feb 20 2013 |
ASJC Scopus subject areas
- Chemistry(all)
- Catalysis
- Biochemistry
- Colloid and Surface Chemistry