title: Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine.
abstract: 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 (Alr) and D-alanine:D-alanine ligase (Ddl). The mechanisms of inhibition of DCS have been well-assessed using Alr and Ddl 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 Mycobacterium tuberculosis Ddl (MtDdl; Rv2981c), and report a kinetic examination of the enzyme with both its native substrate and DCS. MtDdl 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 mm, K(m,D-Ala2) = 3.6 mm). ATP is the first substrate to bind and is necessary for subsequent binding of D-alanine or DCS. The pH dependence of MtDdl kinetic parameters indicate that general base chemistry is involved in the catalytic step. DCS was found to competitively inhibit D-Ala binding at both MtDdl D-Ala sites with equal affinity (K(i,DCS1) = 14 μm, K(i,DCS2) = 25 μm); 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 (pK(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 Ddl-specific inhibitors for use as anti-mycobacterial agents.
authors.affiliation: Mycobacterial Research Division, Medical Research Council National Institute for Medical Research, London, UK.
authors.forename: Gareth A
authors.initials: GA
authors.lastname: Prosser
authors.forename: Luiz Pedro S
authors.initials: LP
authors.lastname: de Carvalho
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000904:Antibiotics, Antitubercular / Q000737:chemistry*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D001667:Binding, Competitive; D003523:Cycloserine / Q000737:chemistry*; D004151:Dipeptides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011485:Protein Binding
chemical_list: D000904:Antibiotics, Antitubercular; D001426:Bacterial Proteins; D004151:Dipeptides; C002956:alanylalanine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003523:Cycloserine; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase; D000409:Alanine