Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

中文翻译：

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人天冬氨酰（天冬酰胺酰）β-羟化酶的活性位点表征和活性。

人天冬氨酰/天冬酰胺酰 β-羟化酶 (HAAH) 是非血红素 Fe2+/α-酮戊二酸 (αKG) 依赖性加氧酶超家族的成员，具有非典型活性位点。HAAH 羟基化表皮生长因子 (EGF) 样结构域以从底物天冬酰胺或天冬氨酸形成 β-羟基化产物，并且已被认为对多种癌症具有负面影响。除了铁，HAAH 还结合二价钙，尽管后者的作用尚不清楚。在此，金属结合化学和对酶稳定性和活性的影响已通过生化和生物物理相结合的方法进行了评估。HAAH 活性位点的金属结合参数通过使用等温滴定量热法测定，除了催化 Fe2+ 辅因子外，还证明了催化结构域中 Ca2+ 的高亲和力调节结合位点。我们分析了各种活性位点衍生物，利用 LC-MS 和新的 HPLC 技术来确定金属结合和第二配位球在酶活性中的作用，发现以前未报告的残基对 HAAH 转换至关重要。对 HAAH 体外生化功能的分析进一步了解了其对细胞生化和代谢途径的重要性。