Purine nucleoside phosphorylase .3. Reversal of purine base specificity by site-directed mutagenesis
Purine nucleoside phosphorylase .3. Reversal of purine base specificity by site-directed mutagenesis
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DOI：
10.1021/bi961971n
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发表时间：
1997-09-30
期刊：
影响因子：
2.9
通讯作者：
Erion, MD
中科院分区：
文献类型：
--
作者：
Stoeckler, JD;Poirot, AF;Erion, MD
人嘌呤核苷磷酸化酶 (PNP) 对 6-氧代嘌呤核苷具有高度特异性，其对肌苷的催化效率 (k(cat)/K-M) 比腺苷高 350000 倍。晶体学研究确定 Asn243 和 Glu201 是主要负责底物特异性的残基。诱变研究的结果表明，两个残基的侧链对于有效催化也是必不可少的 [Erion, M. D., et al. (1997a) 生物化学, 36, 11725-11734]，其他机制研究预测 Asn243 通过向嘌呤碱基 N7 提供氢键来稳定过渡态结构，从而参与催化作用 [Erion, M. D., et al. (1997a) Biochemistry, 36, 11725-11734] (1997b) 生物化学 36, 11735-11748]。为了改变人类 PNP 的底物特异性，设计了 Asn243 和 Glu201 突变体来逆转氢键供体和受体与嘌呤碱基的相互作用。用 Asp 而不是其他氨基酸替代 Asn243，导致腺苷的 k(cat) 增加 5000 倍，总体催化效率增加 4300 倍。此外，Asn243Asp 突变体显示相对于肌苷对腺苷的偏好增加 2.4 倍，底物特异性 (k(cat)/K-M) 相对于野生型改变 800000 倍国民党。双突变体 Asn243Asp::Glu201Gln 相对于野生型 PNP，腺苷的催化效率提高了 190 倍，相对于肌苷，腺苷的偏好性提高了 480 倍，相对于野生型 PNP，腺苷的偏好性比肌苷的偏好性提高了 1.7 x 10(8) 倍。 Asn243Asp 突变体还显示出合成 2,6-二氨基嘌呤核苷的催化效率 (1.4 x 10(6) M-1 s(-1)) 与野生型 PNP 及其天然底物次黄嘌呤和鸟嘌呤处于同一数量级。Asn243Asp 突变体代表了蛋白质工程显着改变底物特异性同时保持高催化效率的例子。
Human purine nucleoside phosphorylase (PNP) is highly specific for 6-oxopurine nucleosides with a catalytic efficiency (k(cat)/K-M) for inosine 350000-fold greater than for adenosine. Crystallographic studies identified Asn243 and Glu201 as the residues largely responsible for the substrate specificity. Results from mutagenesis studies demonstrated that the side chains for both residues were also essential for efficient catalysis [Erion, M. D., et al. (1997a) Biochemistry, 36, 11725-11734], Additional mechanistic studies predicted that Asn243 participated in catalysis by stabilizing the transition state structure through hydrogen bond donation to N7 of the purine base [Erion, M. D., et al. (1997b) Biochemistry 36, 11735-11748]. In an effort to alter the substrate specificity of human PNP, mutants of Asn243 and Glu201 were designed to reverse hydrogen bond donor and acceptor interactions with the purine base. Replacement of Asn243 with Asp, but not with other amino acids, led to a 5000-fold increase in k(cat) for adenosine and a 4300-fold increase in overall catalytic efficiency, Furthermore, the Asn243Asp mutant showed a 2.4-fold preference for adenosine relative to inosine and a 800000-fold change in substrate specificity (k(cat)/K-M) relative to wild-type PNP. The double mutant, Asn243Asp::Glu201Gln, exhibited a 190-fold increase in catalytic efficiency with adenosine relative to wild-type PNP, a 480-fold preference for adenosine relative to inosine, and a 1.7 x 10(8)-fold change in preference for adenosine over inosine relative to wildtype PNP. The Asn243Asp mutant was also shown to synthesize 2,6-diaminopurine riboside with a catalytic efficiency (1.4 x 10(6) M-1 s(-1)) on the same order of magnitude as wild-type PNP with its natural substrates hypoxanthine and guanine, The Asn243Asp mutants represent examples in which protein engineering significantly altered substrate specificity while maintaining high catalytic efficiency.