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 −4 m and 9.7 × 10 −4 m 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 −4 m and 2.1 × 10 −3 m , respectively. The ratio of K m d -alanine to K i d -cycloserine is 0.71, and the ratio of K m l -alanine to K i l -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.