Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site.
Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site.
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工程化胱硫醚-γ-裂解酶的结构快照揭示了静电相互作用在活性位点中的关键作用。
DOI：
10.1021/acs.biochem.6b01172
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Zhang,YanJessie
中科院分区：
文献类型：
--
作者：
Yan,Wupeng;Stone,Everett;Zhang,YanJessie
能够降解蛋氨酸 (l-Met) 的酶疗法引起了人们的极大兴趣，因为许多恶性肿瘤对 l-Met 消耗极其敏感。为了耗尽人血清中的蛋氨酸池，我们之前基于人胱硫醚-γ-裂解酶支架（hCGL-NLV）设计了一种l-Met降解酶，以避免在细菌来源的蛋氨酸-γ-裂解酶的临床前应用中观察到的免疫原性和稳定性问题。为了进一步了解控制 hCGL-NLV 先导分子化学的结构-活性关系，我们进行了一项生物物理表征活动，捕获了 hCGL-NLV 的晶体结构 (2.2 Å) 和不同的反应中间体，包括内部醛亚胺、底物结合、偕二胺和外部醛亚胺形式。奇怪的是，在高盐条件下结晶的 hCGL-NLV 的另一种形式显示出局部未折叠的活性位点，与离子强度函数的活性抑制相关。随后的突变和动力学实验指出，必需辅因子吡哆醛 5'-磷酸 (PLP) 的磷酸盐与残基 R62 之间的盐桥在催化 β- 和 γ- 消除中发挥着重要作用。我们的研究表明，氯化钠等溶剂离子会破坏 R62 和 PLP 之间的静电相互作用，从而降低催化效率。
Enzyme therapeutics that can degradel-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive tol-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered anl-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5′-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.