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) |
|---|---|
| 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 |
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ASJC Scopus subject areas
- Chemistry(all)
- Catalysis
- Biochemistry
- Colloid and Surface Chemistry
Cite this
Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase. / Toney, Michael D.; Castro, Joan Nieto; Addington, Trevor A.
In: Journal of the American Chemical Society, Vol. 135, No. 7, 20.02.2013, p. 2509-2511.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Heavy-enzyme kinetic isotope effects on proton transfer in alanine racemase
AU - Toney, Michael D.
AU - Castro, Joan Nieto
AU - Addington, Trevor A.
PY - 2013/2/20
Y1 - 2013/2/20
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84874050462&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84874050462&partnerID=8YFLogxK
U2 - 10.1021/ja3101243
DO - 10.1021/ja3101243
M3 - Article
C2 - 23373756
AN - SCOPUS:84874050462
VL - 135
SP - 2509
EP - 2511
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 7
ER -