From the Enzyme Biochemistry Group, Max Planck Institute for Biophysical Chemistry, Göttingen D-37077, Germany.
J Biol Chem. 2014 May 9;289(19):12962-75. doi: 10.1074/jbc.M113.545038. Epub 2014 Mar 22.
The structural and functional characterization of human enzymes that are of potential medical and therapeutic interest is of prime significance for translational research. One of the most notable examples of a therapeutic enzyme is L-asparaginase, which has been established as an antileukemic protein drug for more than four decades. Up until now, only bacterial enzymes have been used in therapy despite a plethora of undesired side effects mainly attributed to the bacterial origins of these enzymes. Therefore, the replacement of the currently approved bacterial drugs by human homologs aiming at the elimination of adverse effects is of great importance. Recently, we structurally and biochemically characterized the enzyme human L-asparaginase 3 (hASNase3), which possesses L-asparaginase activity and belongs to the N-terminal nucleophile superfamily of enzymes. Inspired by the necessity for the development of a protein drug of human origin, in the present study, we focused on the characterization of another human L-asparaginase, termed hASNase1. This bacterial-type cytoplasmic L-asparaginase resides in the N-terminal subdomain of an overall 573-residue protein previously reported to function as a lysophospholipase. Our kinetic, mutagenesis, structural modeling, and fluorescence labeling data highlight allosteric features of hASNase1 that are similar to those of its Escherichia coli homolog, EcASNase1. Differential scanning fluorometry and urea denaturation experiments demonstrate the impact of particular mutations on the structural and functional integrity of the L-asparaginase domain and provide a direct comparison of sites critical for the conformational stability of the human and E. coli enzymes.
具有潜在医学和治疗意义的人类酶的结构和功能特征是转化研究的首要任务。治疗酶的一个最著名的例子是 L-天冬酰胺酶，它已经作为一种抗白血病蛋白药物使用了四十多年。到目前为止，尽管这些酶主要归因于细菌起源而存在许多不良副作用，但在治疗中仅使用了细菌酶。因此，用旨在消除不良反应的人类同源物替代目前批准的细菌药物非常重要。最近，我们从结构和生化角度表征了具有 L-天冬酰胺酶活性的酶人类 L-天冬酰胺酶 3（hASNase3），它属于 N-末端亲核体超家族的酶。受开发源自人类的蛋白质药物的必要性的启发，在本研究中，我们专注于另一种人类 L-天冬酰胺酶的表征，称为 hASNase1。这种细菌型细胞质 L-天冬酰胺酶位于先前报道的作为溶血磷脂酶起作用的全长 573 个残基蛋白的 N-末端亚结构域中。我们的动力学、突变、结构建模和荧光标记数据突出了 hASNase1 的变构特征，这些特征类似于其大肠杆菌同源物 EcASNase1 的特征。差示扫描荧光法和脲变性实验证明了特定突变对 L-天冬酰胺酶结构域的结构和功能完整性的影响，并直接比较了对人类和大肠杆菌酶构象稳定性至关重要的位点。
