L-Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys45 residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion-enriched human arginase I mutant (HAI) was Co2+ > Ni2+ ≫ Mn2+. The overall kcat/KM values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40 kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13 mg/kg and Ni-HAI-PEG20L at 15 mg/kg was able to maintain a L-Arg level below its detection limit for over 120 h after one injection. The body weights of mice could return to normal levels within 5 days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. KEY POINTS: • Mono-PEGylation applied on human arginase I mutant (HAI) successfully. • The catalytic efficiency of Co- and Ni-enriched HAI was higher than the wild type. • At least eight types of cancer cell lines were inhibited by Co- and Ni-HAI-PEG20L. • Co- and Ni-HAI-PEG20L were able to achieve weekly depletion of L-Arg. Graphical abstract.

中文翻译：

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一种生物工程化的精氨酸消耗酶，可作为抗癌的长效治疗剂。

L-精氨酸（L-Arg）耗竭在癌症治疗中引起了极大的关注。尽管精氨酸脱亚氨酶（ADI）和人精氨酸酶I两种类型的精氨酸消耗酶正在临床试验中，但聚乙二醇化作用的随机位点，重金属作为辅因子的功效低以及免疫原性限制了这些药物的性能并造成了困难以均匀的生产方式。在这里，我们筛选了十种催化金属离子，并通过将Cys45残基与PEG-马来酰亚胺缀合以最大程度地降低活性降低并产生均一的产物，成功产生了位点特异性单PEG化人精氨酸酶I突变体。富含金属离子的人精氨酸酶I突变体（HAI）的催化效率趋势为Co2 +> Ni2 +≫ Mn2 +。Co-HAI和Ni-HAI的总kcat / KM值分别比Mn-HAI高8.7倍和5.2倍。此外，酶动力学和圆二色性光谱法的结果表明，附着在HAI表面上的20或40 kDa线性和支链PEG不影响酶活性和蛋白质二级结构。体外研究表明，Co-HAI-PEG20L和Ni-HAI-PEG20L均抑制八种癌细胞系的生长。小鼠体内的药效学研究表明，腹腔内给予13 mg / kg的Co-HAI-PEG20L和15 mg / kg的Ni-HAI-PEG20L腹膜内注射后，可以维持L-Arg水平低于其检出限超过120小时一针。注射后5天内，小鼠的体重可能恢复到正常水平，这表明剂量具有良好的耐受性。因此，Ni-HAI-PEG20L和Co-HAI-PEG20L都是有望用于癌症治疗的候选药物。关键点：•单聚乙二醇化成功应用于人精氨酸酶I突变体（HAI）。•富含钴和镍的HAI的催化效率高于野生型。•Co-和Ni-HAI-PEG20L抑制了至少八种癌细胞系。•Co-和Ni-HAI-PEG20L能够每周消耗L-Arg。图形概要。