Department of Biochemistry and Molecular Biology, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97239-3098, USA.
Int J Parasitol. 2011 Apr;41(5):545-52. doi: 10.1016/j.ijpara.2010.12.006. Epub 2011 Jan 11.
Arginase (ARG), the enzyme that catalyzes the conversion of arginine to ornithine and urea, is the first and committed step in polyamine biosynthesis in Leishmania. The creation of a conditionally lethal Δarg null mutant in Leishmania mexicana has established that ARG is an essential enzyme for the promastigote form of the parasite and that the enzyme provides an important defense mechanism for parasite survival in the eukaryotic host. Furthermore, human ARGI (HsARGI) has also been implicated as a key factor in parasite proliferation. Thus, inhibitors of ARG offer a rational paradigm for drug design. To initiate a search for inhibitors of the L. mexicana ARG (LmARG), recombinant LmARG and HsARGI enzymes were purified from Escherichia coli. Both LmARG and HsARGI were specific for l-arginine and exhibited no activity with either d-arginine or agmatine as possible substrates. LmARG exhibited a K(m) of 25±4mM for l-arginine, a pH optimum ∼9.0, and was dependent upon the presence of a divalent cation, preferentially manganese. A K(m) of 13.5 ± 2mM for l-arginine was calculated for the HsARGI. A collection of 37 compounds was evaluated against both enzymes. Twelve of these compounds were identified as being either strong inhibitors of both LmARG and HsARGI or differential inhibitors between the two enzymes. Of the 12 compounds, six were selected for further analysis and the type and extent of inhibition determined.
精氨酸酶（ARG）是一种催化精氨酸转化为鸟氨酸和尿素的酶，是莱姆希曼原虫中多胺生物合成的第一步和关键步骤。在莱姆希曼原虫中创建条件致死的Δarg 缺失突变体，证明了 ARG 是寄生虫前鞭毛体形式的必需酶，并且该酶为寄生虫在真核宿主中的存活提供了重要的防御机制。此外，人 ARGI（HsARGI）也被认为是寄生虫增殖的关键因素。因此，ARG 的抑制剂为药物设计提供了合理的范例。为了开始寻找莱姆希曼原虫 ARG（LmARG）的抑制剂，从大肠杆菌中纯化了重组 LmARG 和 HsARGI 酶。LmARG 和 HsARGI 均特异性识别 l-精氨酸，并且没有活性与 d-精氨酸或胍丁胺作为可能的底物。LmARG 对 l-精氨酸的 K(m)为 25±4mM，pH 最佳值约为 9.0，并且依赖于二价阳离子的存在，优选锰。计算出 HsARGI 对 l-精氨酸的 K(m)为 13.5 ± 2mM。评估了 37 种化合物对两种酶的抑制作用。其中 12 种化合物被鉴定为对 LmARG 和 HsARGI 均具有强抑制作用，或对两种酶具有差异抑制作用。在 12 种化合物中，选择了 6 种进行进一步分析，并确定了抑制类型和程度。
