Mechanism of d-Cycloserine Action: Alanine Racemase from Escherichia coli W1

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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 Vmax 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 (Ki) of 6.5 × 10−4m and 2.1 × 10−3m, respectively. The ratio of Kmd-alanine to Kid-cycloserine is 0.71, and the ratio of Kml-alanine to Kil-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.

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