Kinetic mechanism and inhibition of M ycobacterium tuberculosis d ‐alanine: d ‐alanine ligase by the antibiotic d ‐cycloserine

d ‐cycloserine ( DCS ) is an antibiotic that is currently used in second‐line treatment of tuberculosis. DCS is a structural analogue of d ‐alanine, and targets two enzymes involved in the cytosolic stages of peptidoglycan synthesis: alanine racemase ( A lr) and d ‐alanine: d ‐alanine ligase ( D dl). The mechanisms of inhibition of DCS have been well‐assessed using A lr and D dl enzymes from various bacterial species, but little is known regarding the interactions of DCS with the mycobacterial orthologues of these enzymes. We have over‐expressed and purified recombinant M ycobacterium tuberculosis D dl ( M t D dl; R v2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS . M t D dl is activated by K + , follows an ordered ter ter mechanism and displays distinct affinities for d ‐Ala at each d ‐Ala binding site ( K m, d ‐Ala1  = 0.075 m m , K m, d ‐Ala2  = 3.6 m m ). ATP is the first substrate to bind and is necessary for subsequent binding of d ‐alanine or DCS . The pH dependence of M t D dl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit d ‐Ala binding at both M t D dl d ‐ A la sites with equal affinity ( K i,DCS1  = 14 μ m , K i,DCS2  = 25 μ m ); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of K i,DCS2 revealed a loss of DCS binding affinity at high pH (p K a  = 7.5), suggesting that DCS binds optimally in the zwitterionic form. The results of this study may assist in the design and development of novel D dl‐specific inhibitors for use as anti‐mycobacterial agents.