The lysine residue binding with the cofactor pyridoxal 5-phosphate (PLP) plays an important role in catalysis, such as in the transaldimination and abstraction of ␣-hydrogen from a substrate amino acid in PLP-dependent enzymes. We studied the role of Lys 39 of alanine racemase (EC 5.1.1.1) from Bacillus stearothermophilus, the PLP-binding residue of the enzyme, by replacing it site-specifically with alanine and characterizing the resultant K39A mutant enzyme. The mutant enzyme turned out to be inherently inactive, but gained an activity as high as about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine. The amineassisted activity of the mutant enzyme depended on the pK a values and molecular volumes of the alkylamines used. A strong kinetic isotope effect was observed when ␣-deuterated D-alanine was used as a substrate in the methylamine-assisted reaction, but little effect was observed using its antipode. In marked contrast, only Lenantiomer of alanine showed a solvent isotope effect in deuterium oxide in the methylamine-assisted reaction. These results suggest that methylamine serves as a base not only to abstract the ␣-hydrogen from D-alanine but also to transfer a proton from water to the ␣-position of the deprotonated (achiral) intermediate to form D-alanine. Therefore, the exogenous amine can be regarded as a functional group fully representing Lys 39 of the wild-type enzyme. Lys 39 of the wild-type enzyme probably acts as the base catalyst specific to the D-enantiomer of alanine. Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the K39A mutant.