Mechanism of d -Cycloserine Action: Alanine Racemase from Escherichia coli W

Abstract

The antibiotic d-cycloserine is an effective inhibitor of alanine racemase. The lack of inhibition by l-cycloserine of alanine racemase from Staphylococcus aureus led Roze and Strominger to formulate the cycloserine hypothesis. This hypothesis states that d-cycloserine has the conformation required of the substrates on the enzyme surface and that l-cycloserine cannot have this conformation. Alanine racemase from Escherichia coli W has been examined to establish whether these observations are a general feature of all alanine racemases. The enzyme (molecular weight = 95,000) has Michaelis-Menten constants of 4.6 × 10⁻⁴m and 9.7 × 10⁻⁴m for d- and l-alanine, respectively. The ratio of V_max in the d- to l-direction is 2.3. The equilibrium constant calculated from the Haldane relationship is 1.11 ± 0.15. Both d- and l-cycloserine are competitive inhibitors with constants (K_i) of 6.5 × 10⁻⁴m and 2.1 × 10⁻³m, respectively. The ratio of K_md-alanine to K_id-cycloserine is 0.71, and the ratio of K_ml-alanine to K_il-cycloserine is 0.46. Since l-cycloserine is an effective inhibitor, it is concluded that the cycloserine hypothesis does not apply to the enzyme from E. coli W.