| Abstract
| - Uridine diphosphateN-acetylmuramate:l-alanine ligase (EC 6.3.2.8,UNAM:l-Ala ligase orMurC gene product) adds the first amino acid to the sugarmoiety of the peptidoglycan precursor, catalyzingone of the essential steps in cell wall biosynthesis for bothgram-positive and gram-negative bacteria.Here, we report our studies on the secondary and quaternarystructures of UNAM:l-Ala ligase fromEscherichia coli. The molecular weight of the purifiedrecombinant enzyme determined by electrosprayionization mass spectrometry agreed well with the molecular weightdeduced from the DNA sequence.Through sedimentation equilibrium analysis, we show that theenzyme exists in equilibrium betweenmonomeric and dimeric forms and that the dissociation constant of thedimer, Kd, was determined to be1.1 ± 0.4 μM at 37 °C and 0.58 ± 0.30 μM at 4 °C. Avery similar Kd value was also obtained at37°C by gel filtration chromatography. The secondary structure ofthe enzyme was characterized by circulardichroism spectroscopy. No change in the secondary structure wasobserved between the monomericand dimeric forms of the enzyme. The activity assays at enzymeconcentrations both below and abovethe determined Kd value lead to the conclusionthat the enzyme is active both as dimers and as monomersand that the specific activity is independent of the oligomerizationstate.
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