Alanine racemase (ALR) converts L-alanine to D-alanine, a vital component of the bacterial cell wall. Consequently, ALR is an interesting target for antibiotic research. In <i>Mycobacterium</i> tuberculosis (MT), the causative agent of tuberculosis, active ALR exists as a dimer, with each dimer possessing two active sites, each formed on the interface between the individual monomers. A complete understanding of the interaction between the two monomers could provide insight into approaches to inactivate the dimeric ALR. We investigated the degree of association of ALR in a variety of buffers by means of high performance size exclusion liquid chromatography (HPSEC), In phosphate, tris, bicine, and glycine buffers over the pH range of 5-12, MT ALR eluted with a retention volume consistent with a molar mass of approximately 70 kDa, i.e., as a monomer-dimer equilibrium mixture. However, in pH 8.0 borate buffer, MT ALR eluted with a retention volume consistent with a molar mass of 80 kDa, i.e., the expected dimer. Further, as the pH of the borate buffer increased, the HPSEC retention volume of MT ALR decreased. At pH 10.5, MT ALR eluted with a retention volume consistent with a molar mass of approximately 130 kDa, i.e., most likely as a dimer-tetramer equilibrium mixture. These results are consistent with MT ALR possessing a phosphate binding-site that inhibits the dimerization.

Oligomeric state of <i>Mycobacterium tuberculosis</i>’ alanine racemase is highly dependent on buffering ion as well as pH

254th National Meeting (2017)

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Oligomeric state of Mycobacterium tuberculosis’ alanine racemase is highly dependent on buffering ion as well as pH