Binding Affinity Determines Substrate Specificity and Enables Discovery of Substrates for N-Myristoyltransferases.
Binding Affinity Determines Substrate Specificity and Enables Discovery of Substrates for N-Myristoyltransferases.
复制标题
DOI：
10.1021/acscatal.1c03330
复制
发表时间：
2021-12-17
期刊：
影响因子：
12.9
通讯作者：
Lin H
中科院分区：
文献类型：
--
作者：
Su D;Kosciuk T;Yang M;Price IR;Lin H
由Michaelis-Menten方程导出的动力学参数kcat和Km被广泛用于酶的表征。kcat/Km被认为是酶对其底物的催化效率或底物特异性。n -肉豆蔻酰基转移酶（NMTs）催化许多真核蛋白的n端甘氨酸肉豆蔻酰基化。令人惊讶的是，我们在体外发现人类NMT1可以接受乙酰辅酶a并催化乙酰化，其kcat和Km值与肉豆蔻酰化相似。然而，当反应中同时存在乙酰辅酶a和肉豆蔻酰基辅酶a时，NMT1几乎只催化肉豆蔻酰化。这种现象是由于NMT1对肉豆蔻酰基辅酶a和乙酰辅酶a的结合亲和力差异很大（估计Kd分别为14.7 nM和10.1 μM）。当两者都存在时，NMT1基本上完全与肉豆蔻酰基辅酶a结合，因此只催化肉豆蔻酰化。NMT1的例子强调了结合亲和力在确定酶的底物特异性方面的关键作用，这与酶学中传统的底物特异性由kcat/Km值定义的观点相反。这种理解很容易解释大量生物学文献显示酶-底物对的共免疫沉淀，这些酶催化蛋白质翻译后修饰（PTM），包括磷酸化、乙酰化和泛素化。此外，这种理解允许通过鉴定PTM酶的相互作用蛋白来发现底物蛋白，我们通过挖掘可用的相互作用组数据鉴定了人类NMT1/2的三个以前未知的底物蛋白（LRATD1， LRATD2和ERICH5）来证明这一点。
Kinetic parameters (kcat and Km) derived from the Michaelis–Menten equation are widely used to characterize enzymes. kcat/Km is considered the catalytic efficiency or substrate specificity of an enzyme toward its substrate. N-Myristoyltransferases (NMTs) catalyze the N-terminal glycine myristoylation of numerous eukaryotic proteins. Surprisingly, we find that in vitro human NMT1 can accept acetyl-CoA and catalyze acetylation with kcat and Km values similar to that of myristoylation. However, when both acetyl-CoA and myristoyl-CoA are present in the reaction, NMT1 catalyzes almost exclusively myristoylation. This phenomenon is caused by the dramatically different binding affinities of NMT1 for myristoyl-CoA and acetyl-CoA (estimated Kd of 14.7 nM and 10.1 μM, respectively). When both are present, NMT1 is essentially entirely bound by myristoyl-CoA and thus catalyzes myristoylation exclusively. The NMT1 example highlights the crucial role of binding affinity in determining the substrate specificity of enzymes, which in contrast to the traditionally held view in enzymology that the substrate specificity is defined by kcat/Km values. This understanding readily explains the vast biological literature showing the coimmunoprecipitation of enzyme–substrate pairs for enzymes that catalyzes protein post-translational modifications (PTM), including phosphorylation, acetylation, and ubiquitination. Furthermore, this understanding allows the discovery of substrate proteins by identifying the interacting proteins of PTM enzymes, which we demonstrate by identifying three previously unknown substrate proteins (LRATD1, LRATD2, and ERICH5) of human NMT1/2 by mining available interactome data.
登录
查看更多内容
影响因子：
16.6
作者：
Dian, Cyril;Perez-Dorado, Inmaculada;Giglione, Carmela
通讯作者：
Giglione, Carmela
DOI：
10.1073/pnas.97.1.179
发表时间：
2000-01-04
影响因子：
11.1
作者：
Pandey, A;Podtelejnikov, AV;Lodish, HF
通讯作者：
Lodish, HF
影响因子：
16.6
作者：
Thinon, Emmanuelle;Serwa, Remigiusz A.;Broncel, Malgorzata;Brannigan, James A.;Brassat, Ute;Wright, Megan H.;Heal, William P.;Wilkinson, Anthony J.;Mann, David J.;Tate, Edward W.
通讯作者：
Tate, Edward W.
影响因子：
4
作者：
Kosciuk T;Lin H
通讯作者：
Lin H
影响因子：
2.9
作者：
Farazi, TA;Waksman, G;Gordon, JI
通讯作者：
Gordon, JI