Biosynthesis of Uridine Diphospho-N-acetylmuramic Acid

Phosphoenolpyruvate : uridine -5' -diphospho -N -acetyl -2amino-2-deoxyglucose-3-enolpyruvyltransferase, an enzyme which transfers enolpyruvate from phosphoenolpyruvate to uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) with the liberation of inorganic orthophosphate was purified 326-fold from En2erobacter cloacae NRC 492.The transferase was free of UDP-GlcNAc-enolpyruvate reductase, an enzyme which catalyzes the reduction of UDP-GlcNAcenolpyruvate to form uridine-5'-diphospho-N-acetyhnuramic acid.The enzyme was specific for the indicated substrates, and the optimum pH range for the reaction was from pH 6.7 to pH 7.4.The reaction was reversible as judged by radioactive inorganic orthophosphate exchange.The K, for phosphoenolpyruvate was 3 x 10y5 M and that for UDP-GlcNAc was 4.6 x 10u4 M. The available data suggest that the enzyme requires thiol group(s) for its activity.The energy of activation of the enzyme, calculated from the Arrhenius plot, was 15 kcal per mole.Pure UDP-GlcNAcenolpyruvate has been prepared in milligram quantities.The hydrolysis of this nucleotide in 1 M HCl at 100" liberated 1 mole of pyruvate per mole of uridine.Hydrogenation of the nucleotide followed by hydrolysis in 6 N HCl at 100" resulted in the liberation of muramic acid.On the basis of its chemical properties and infrared and ultraviolet absorption studies, the nucleotide has been characterized as uridine-S'diphospho-N-acetyl-2-amino-2-deoxy-3 -enolpyruvylglucose.Infrared and ultraviolet absorption spectra are given.In a previous paper (l), we reported a pathway for the biosynthesis of uridine-5'-diphospho-N-acetylmuramic acid.