Li Zhimin, Kulakova Liudmila, Li Ling, Galkin Andrey, Zhao Zhiming, Nash Theodore E, Mariano Patrick S, Herzberg Osnat, Dunaway-Mariano Debra
Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131, USA.
Bioorg Chem. 2009 Oct;37(5):149-61. doi: 10.1016/j.bioorg.2009.06.001. Epub 2009 Jun 13.
Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an insilico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N(alpha)-benzoyl-l-arginine, and N(omega)-amino-l-arginine as substrates but not agmatine, l-homoarginine, N(alpha)-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424-alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k(1)=80s(-1)) and hydrolysis (k(2)=35s(-1)) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k(cat)=2.6s(-1)), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N(omega)-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.
本文的主题——蓝氏贾第鞭毛虫精氨酸脱亚氨酶（GlAD），属于胍基修饰酶超家族的水解酶分支，该家族成员利用半胱氨酸介导的亲核催化作用促进L-精氨酸及其天然衍生物的脱亚氨基反应。蓝氏贾第鞭毛虫是人类贾第虫病的病原体。本文报道的RNA干扰/反义RNA基因沉默研究结果表明，GlAD对蓝氏贾第鞭毛虫滋养体的存活至关重要，因此是开发治疗贾第虫病治疗药物的潜在靶点。在大肠杆菌中制备了同二聚体重组蛋白，用于深入的生化特性研究。具有两个结构域的GlAD单体由一个N端结构域和一个C端结构域组成，N端结构域与其细菌AD对应物具有相同的活性位点结构（由计算机模拟模型描绘）和动力学特性（通过稳态和瞬态动力学分析确定），C端结构域的折叠和功能未知。研究发现GlAD在很宽的pH范围内都有活性，并且可以接受L-精氨酸、L-精氨酸乙酯、N（α）-苯甲酰-L-精氨酸和N（ω）-氨基-L-精氨酸作为底物，但不能接受胍丁胺、L-高精氨酸、N（α）-苯甲酰-L-精氨酸乙酯或多种含精氨酸的肽。证明了半胱氨酸424-烷基硫脲离子共价酶加合物的中间体，并测定了中间体形成（k(1)=80s(-1)）和水解（k(2)=35s(-1)）的速率常数。稳态速率常数（k(cat)=2.6s(-1)）相对较低，表明瓜氨酸形成后的步骤是限速步骤。简要探讨了使用半胱氨酸定向试剂对GlAD的抑制作用。S-亚硝基-L-高半胱氨酸被证明是一种活性位点定向的不可逆抑制剂，而N（ω）-氰基-L-精氨酸不抑制GlAD，反而被证明是蜡样芽孢杆菌AD的活性位点定向的不可逆抑制剂。
