Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.
Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.
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计算机模拟揭示了天然和突变的人嘌呤核苷磷酸化酶中糖苷键裂解的底物特异性。
DOI：
10.1021/acs.biochem.5b01347
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发表时间：
2016
期刊：
影响因子：
2.9
通讯作者：
B. Brandsdal
中科院分区：
文献类型：
--
作者：
G. Isaksen;K. H. Hopmann;J. Åqvist;B. Brandsdal
嘌呤核苷磷酸化酶（PNP）催化嘌呤核糖核苷和2′-脱氧核糖核苷的可逆磷酸化，生成嘌呤碱基和（2′-脱氧）核糖1-磷酸。虽然这种酶已被广泛研究，但有关其催化机制的几个问题在很大程度上仍未得到解答。利用密度泛函理论计算和广泛的经验价键（EVB）模拟，阐明了磷酸基和关键氨基酸残基在催化反应中的作用以及嘌呤环的质子化状态。腺苷、肌苷和鸟苷的自由能表面与从头算数据拟合，当表面用于模拟相应的酶反应时，所得的定量结果与实验数据一致。同源底物6-氨基嘌呤（肌苷和鸟苷）通过广泛的氢键与PNP相互作用，但发现底物特异性是沿反应坐标的静电预组织能的直接结果。Asn243先前被鉴定为提供底物特异性的关键残基。Asn243突变为Asp对底物特异性有显著影响，使6-氨基嘌呤和6-氧嘌呤同样可以作为底物。这种特殊突变的主要影响是原生酶和Asn243Asp突变体之间静电预组织能的变化，明显倾向于腺苷而不是肌苷和鸟苷。因此，EVB模拟表明，这种特殊的突变影响活性位点的静电预组织，这反过来可以解释底物特异性。
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.
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影响因子：
3.4
作者：
Saen-Oon, Suwipa;Ghanem, Mahmoud;Schwartz, Steven D.
通讯作者：
Schwartz, Steven D.
DOI：
10.2210/pdb2pnp/pdb
发表时间：
1992-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
S. Ealick;S. Rule;D. Carter;T. Greenhough;Y. Babu;W. Cook;J. Habash;J. Helliwell;J. Stoeckler;R. Parks
通讯作者：
S. Ealick;S. Rule;D. Carter;T. Greenhough;Y. Babu;W. Cook;J. Habash;J. Helliwell;J. Stoeckler;R. Parks
通过 19F-NMR 和晶体学测定人 PNP 中的催化位点构象。
影响因子：
--
作者：
Suarez,Javier;Haapalainen,AnttiM;Cahill,SeanM;Ho,Meng-Chiao;Yan,Funing;Almo,StevenC;Schramm,VernL
通讯作者：
Schramm,VernL
影响因子：
2.9
作者：
H. Deng;S. Cahill;J. Abad;A. Lewandowicz;R. Callender;V. Schramm;Roger A. Jones
通讯作者：
H. Deng;S. Cahill;J. Abad;A. Lewandowicz;R. Callender;V. Schramm;Roger A. Jones
影响因子：
2.9
作者：
Fedorov, A;Shi, W;Almo, SC
通讯作者：
Almo, SC