Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine
Kinetic mechanism and inhibition of Mycobacterium tuberculosis D-alanine:D-alanine ligase by the antibiotic D-cycloserine
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DOI：
10.1111/febs.12108
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发表时间：
2013-02-01
期刊：
影响因子：
5.4
通讯作者：
de Carvalho, Luiz Pedro S.
中科院分区：
文献类型：
--
作者：
Prosser, Gareth A.;de Carvalho, Luiz Pedro S.
d-环丝氨酸（DCS）是一种抗生素，目前用于结核病的二线治疗。DCS是d-丙氨酸的结构类似物，靶向两种参与肽聚糖合成的胞质阶段的酶：丙氨酸消旋酶（Alr）和d-丙氨酸：d-丙氨酸连接酶（Ddl）。利用来自不同细菌种类的Alr和Ddl酶，已经对DCS的抑制机制进行了很好的评估，但关于DCS与这些酶的分枝杆菌同源物的相互作用知之甚少。我们过度表达和纯化了重组结核分枝杆菌Ddl (MtDdl; Rv2981c)，并报道了该酶与天然底物和DCS的动力学检测。MtDdl被K+激活，遵循有序的ter机制，在每个d-Ala结合位点显示出不同的亲和力（Km,d-Ala1=0.075mm, Km,d-Ala2= 3.6mm）。ATP是第一个结合的底物，是随后结合d-丙氨酸或DCS所必需的。MtDdl动力学参数的pH依赖性表明，一般碱化学参与了催化步骤。发现DCS在两个MtDdl d-Ala位点竞争性地抑制d-Ala结合，具有相同的亲和力（Ki,DCS1=14m, Ki,DCS2=25m）；然而，每个酶活性位点在给定时间内只能容纳单个DCS分子。Ki、DCS2的pH依赖性表明，在高pH （pKa=7.5）下，DCS的结合亲和力丧失，表明DCS以两性离子形式结合最为理想。这项研究的结果可能有助于设计和开发新的ddl特异性抑制剂作为抗分枝杆菌药物。
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 Mycobacteriumtuberculosis 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 (Km,d-Ala1=0.075mm, Km,d-Ala2= 3.6mm). 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 (Ki,DCS1=14m, Ki,DCS2=25m); however, each enzyme active site can only accommodate a single DCS molecule at a given time. The pH dependence of Ki,DCS2 revealed a loss of DCS binding affinity at high pH (pKa=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.