Mechanism of D-cycloserine action: alanine racemase from Escherichia coli W.
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 x 10(-4)m and 9.7 x 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 x 10(-4)m and 2.1 x 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.
D000409:Alanine / Q000378:metabolism; D000645:Ammonium Sulfate; D000440:Butanols; D002247:Carbon Isotopes; D002458:Cell Fractionation; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003523:Cycloserine / Q000494:pharmacology*; D004589:Electrophoresis, Disc; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011479:Protamines; D013237:Stereoisomerism; D013395:Sucrose; D013431:Sulfates
D000440:Butanols; D002247:Carbon Isotopes; D011479:Protamines; D013431:Sulfates; D013395:Sucrose; D003523:Cycloserine; D007535:Isomerases; D000409:Alanine; D000645:Ammonium Sulfate