第四纪MutT-M2 + -AMPCPP-M2 +解决方案结构的复杂和pyrophosphohydrolase行动机制。
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林J, Abeygunawardana C,弗里克DN, Bessman MJ, Mildvan
第四纪MutT-M2 + -AMPCPP-M2 +解决方案结构的复杂和pyrophosphohydrolase行动机制。
生物化学,1997年2月11日,36 (6):1199 - 211。
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9063868 (在PubMed]
- 文摘
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杂种狗酶(129残留物)催化水解的核苷三磷酸腺苷(NTP)替换很少攻击βp,收益率NMP和焦磷酸。它需要两个二价阳离子,形成一个活跃的E-M2 + -NTP-M2 +复杂。自由酶的溶液结构由five-stranded混合β褶板连接循环I-alpha-helix i循环II,两个急转弯和循环III和终止循环IV-alpha-helix II [Abeygunawardana C。et al。(1995)生物化学34岁,14997 - 15005]。作业的骨干15 n和NH共振和15 n和侧链氨基共振的第四纪复杂是由1 h-15n HSQC滴定的自由酶MgCl2紧随其后的克分子数相等的AMPCPP / MgCl2。H(α)的任务是由1 h-15n 3 d TOCSY HSQC,和1 h-13c CT-HSQC光谱和骨干和侧链1 H和13 c作业是由3 d一家TOCSY实验。配体所捐赠的蛋白质enzyme-bound二价阳离子,二氧化碳被顺磁的影响H +和Mn2 + CO (C)光谱,Glu-56羧酸盐组,-57年和-98年的酰胺羰基Gly-38。结构复杂的解决方案是计算XPLOR使用2168 NOE和φ83限制蛋白质,11分子内不绑定Mg2 + AMPCPP 22之间的分子间不小狗AMPCPP,距离enzyme-bound Co2 +三个磷原子的二氧化碳+ (NH3) 4 AMPCPP从顺磁的影响Co2 + T1值。小狗的折叠酶的复杂非常类似于自由酶,与小金属和底物结合位点的变化。腺嘌呤环结合疏水裂,与Leu-4交互和Ile-6 beta-strand和ile - 80在6-NH2 beta-strand d组的侧链氨基腺嘌呤方法asn - 119。这不利作用强烈的绑定的杂种狗鸟嘌呤核苷酸,6-keto组。 The ribose binds with its hydroxyl groups oriented toward the solvent and its hydrophobic face interacting with Leu-4, Ile-6, and the gamma-CH2 of Lys-39 of loop I. The metal-triphosphate moiety appears to bind in the second coordination sphere of the enzyme-bound divalent cation. One of two intervening water ligands is well positioned to attack P(beta) with inversion and to donate a hydrogen bond to the conserved residue, Glu-53, which may deprotonate or orient the attacking water ligand. Lys-39 which is positioned to interact electrostatically with the alpha-phosphoryl group may facilitate the departure of the leaving NMP. On the basis of the structure of the quaternary complex, a mechanism of the MutT reaction is proposed which is qualitatively and quantitatively consistent with kinetic and mutagenesis studies. It is suggested that similar mechanisms may be operative for other enzymes that catalyze substitution at P(beta) of NTP substrates.