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首页> 外文期刊>Journal of Molecular Biology >Elucidation of the specific function of the conserved threonine triad responsible for human l-Asparaginase autocleavage and substrate hydrolysis
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Elucidation of the specific function of the conserved threonine triad responsible for human l-Asparaginase autocleavage and substrate hydrolysis

机译：阐明保守的苏氨酸三合会负责人L-天冬酰胺酶自闭症和底物水解的特定功能

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Our long-term goal is the design of a human l-Asparaginase (hASNase3) variant, suitable for use in cancer therapy without the immunogenicity problems associated with the currently used bacterial enzymes. Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. The key property allowing for the depletion of blood asparagine by bacterial asparaginases is their low micromolar KM value. In contrast, human enzymes have a millimolar KM for asparagine. Toward the goal of engineering an hASNase3 variant with micromolar KM, we conducted a structure/function analysis of the conserved catalytic threonine triad of this human enzyme. As a member of the N-terminal nucleophile family, to become enzymatically active, hASNase3 must undergo autocleavage between residues Gly167 and Thr168. To determine the individual contribution of each of the three conserved active-site threonines (threonine triad Thr168, Thr186, Thr219) for the enzyme-Activating autocleavage and asparaginase reactions, we prepared the T168S, T186V and T219A/V mutants. These mutants were tested for their ability to cleave and to catalyze asparagine hydrolysis, in addition to being examined structurally. We also elucidated the first N-terminal nucleophile plant-type asparaginase structure in the covalent intermediate state. Our studies indicate that, while not all triad threonines are required for the cleavage reaction, all are essential for the asparaginase activity. The increased understanding of hASNase3 function resulting from these studies reveals the key regions that govern cleavage and the asparaginase reaction, which may inform the design of variants that attain a low KM for asparagine.
机译：我们的长期目标是人L-天冬酰胺酶（HASNase3）变体的设计，适用于癌症治疗而没有与目前使用的细菌酶相关的免疫原性问题。天冬酰胺酶催化氨基酸天冬酰胺的水解至天冬氨酸和氨。允许通过细菌天冬酰胺酶耗尽血享生酶的关键特性是它们的低微摩尔km值。相比之下，人酶为天冬酰胺有毫米摩尔克。朝着用微摩尔KM工程的碱酶3变体的目标，我们进行了这种人酶的保守催化苏氨酸三合会的结构/功能分析。作为N-末端亲核官员的成员，为了酶活性，HASNase3必须在残留物组合167和THR168之间进行高沉淀。为了确定三个保守的活性位点苏氨酸（Threonine Triad Thr168，Thr186，Thr219）的个体贡献，用于酶活性的酶的自闭症和天冬酰胺酶反应，我们制备了T168S，T186V和T219A / V突变体。除了在结构上检查之外，还测试这些突变体的能力并催化天冬酰胺水解。我们还阐明了在共价中间状态下的第一n末端亲核试剂型天酰胺酶结构。我们的研究表明，虽然并非所有三合会苏氨酸都是裂解反应所必需的，但所有的副淀粉蛋白活性都是必不可少的。这些研究产生的Hasnase3函数的提高揭示了治理裂解和天冬酰胺酶反应的关键区域，这可能会通知达乙酰胺达到低km的变体的设计。

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