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

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 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.

Figure 1

Michaelis-Menten kinetics for ^HAR (□) and ^DAR (■) with L-alanine (left) and D-alanine (right) (pH 8.9, 25 °C).

Figure 2

Michaelis-Menten kinetics for ^HAR (□) and ^DAR (■) with [2-²H]-L-alanine (left) and [2-²H]-D-alanine (right) (pH 8.9, 25 °C).

Scheme 1