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首页> 外文期刊>Biochemistry >Formiminoglutamase from Trypanosoma Cruzi Is An Arginase-Like Manganese Metalloenzyme
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Formiminoglutamase from Trypanosoma Cruzi Is An Arginase-Like Manganese Metalloenzyme

机译：锥虫锥虫的谷氨酰胺酶是一种类似于精氨酸的锰金属酶

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The crystal structure of formiminoglutamase from Trypanosoma cruzi (TcFIGase) is reported at 1.85 ? resolution. Although the structure of this enzyme was previously determined by the Structural Genomics of Pathogenic Protozoa Consortium (PDB accession code 2A0M), this structure was determined at low pH and lacked bound metal ions; accordingly, the protein was simply annotated as "arginase superfamily protein" with undetermined function. We show that reconstitution of this protein with Mn~(2+) confers maximal catalytic activity in the hydrolysis of formiminoglutamate to yield glutamate and formamide, thereby demonstrating that this protein is a metal-dependent formiminoglutamase. Equilibration of TcFIGase crystals with MnCl_2 at higher pH yields a binuclear manganese cluster similar to that observed in arginase, except that the histidine ligand to the Mn~(2+)_A ion of arginase is an asparagine ligand (N114) to the Mn~(2+)_A ion of TcFIGase. The crystal structure of N114H TcFIGase reveals a binuclear manganese cluster essentially identical to that of arginase, but the mutant exhibits a modest 35% loss of catalytic efficiency (k_(cat)/K_M). Interestingly, when TcFIGase is prepared and crystallized in the absence of reducing agents at low pH, a disulfide linkage forms between C35 and C242 in the active site. When reconstituted with Mn~(2+) at higher pH, this oxidized enzyme exhibits a modest 33% loss of catalytic efficiency. Structure determinations of the metal-free and metal-bound forms of oxidized TcFIGase reveal that although disulfide formation constricts the main entrance to the active site, other structural changes open alternative channels to the active site that may help sustain catalytic activity.
机译：据报道，克氏锥虫的甲酰胺谷氨酰胺酶的晶体结构（TcFIGase）为1.85？解析度。尽管该酶的结构先前是由致病性原生动物联盟的结构基因组学确定的（PDB登录号2A0M），但该结构是在低pH值且缺乏结合的金属离子的情况下确定的。因此，该蛋白质被简单地标注为功能不确定的“精氨酸酶超家族蛋白质”。我们显示该蛋白质与Mn〜（2+）的重组赋予了最大的催化活性，以水解甲米谷氨酸产生谷氨酸和甲酰胺，从而证明该蛋白是金属依赖性的甲米谷氨酰胺酶。在较高pH下用MnCl_2平衡TcFIGase晶体产生的双核锰簇与精氨酸酶中观察到的相似，不同之处在于精氨酸酶Mn〜（2 +）_ A离子的组氨酸配体是Mn〜（ TcFIGase的2 +）_ A离子。 N114H TcFIGase的晶体结构显示出与精氨酸酶基本相同的双核锰簇，但该突变体显示出适度的35％的催化效率损失（k_（cat）/ K_M）。有趣的是，当制备TcFIGase并在低pH下不存在还原剂的情况下进行结晶时，在活性位点C35和C242之间形成了二硫键。当在较高pH下用Mn〜（2+）重构时，这种氧化酶表现出适度的33％的催化效率损失。对无金属和金属结合形式的氧化TcFIGase的结构测定表明，尽管二硫化物的形成限制了活性位点的主要入口，但其他结构变化为活性位点打开了替代通道，可帮助维持催化活性。

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