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.
Department of Chemistry, University of California-Davis, One Shields Avenue, Davis, California 95616, USA. mdtoney@ucdavis.edu
Michael D
MD
Toney
Joan Nieto
JN
Castro
Trevor A
TA
Addington
D000411:Alanine Racemase / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D015394:Molecular Structure; D011522:Protons
D011522:Protons; D003903:Deuterium; D000411:Alanine Racemase