Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme.
Mechanistic study of protein phosphatase-1 (PP1), a catalytically promiscuous enzyme.
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DOI：
10.1021/ja803612z
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发表时间：
2008-10-15
影响因子：
15
通讯作者：
Williams, Nicholas H.
中科院分区：
文献类型：
--
作者：
McWhirter, Claire;Lund, Elizabeth A.;Tanifum, Eric A.;Feng, Guoqiang;Sheikh, Qaiser I.;Hengge, Alvan C.;Williams, Nicholas H.
蛋白质磷酸酶 1 (PP1) 催化的反应已通过线性自由能关系和动力学同位素效应进行了检查。使用底物磷酸 4-硝基苯酯 (4NPP)，反应呈现出钟形 kcat/KM 的 pH 速率曲线，表明酸性和碱性残基均具有催化作用，动力学 pKa 分别为 6.0 和 7.2。一系列芳基单酯底物的酶水解产生的 Brønsted βlg 为 -0.32，比单酯二价阴离子的非催化水解 (-1.23) 的负值要小得多。底物 4NPP 的离去基团中的动力学同位素效应为 18(V/K)bridge = 1.0170 和 15(V/K) = 1.0010，与具有或不具有一般酸催化的其他酶 KIE 相比，与离去基团部分中和的松散过渡态一致。 PP1 还可以有效催化 4-硝基苯基甲基膦酸酯 (4NPMP) 的水解。一系列甲基膦酸芳基酯底物的酶水解产生 -0.30 的 Brønsted βlg，小于碱性水解 (-0.69)，并且与单酯底物测量的 βlg 相似，表明具有相似的过渡态。 KIE 和 βlg 数据表明 4NPMP 碱性水解的过渡态与具有相同离去基团的二酯的过渡态相似。对于 4NPMP 的酶促反应，KIE 表明过渡态比碱性水解反应稍微松散，并且类似于 PP1 催化的单酯反应。数据累积表明芳基磷酸单酯和芳基甲基膦酸酯水解反应的酶促过渡态比两种底物的非酶促水解反应彼此更加相似。
The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKas of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted βlg of -0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (-1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are 18(V/K)bridge = 1.0170 and 15(V/K) = 1.0010 which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Brønsted βlg of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the βlg measured for monoester substrates, indicative of similar transition states. The KIEs and the βlg data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction, and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.
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影响因子：
15
作者：
Cassano, AG;Anderson, VE;Harris, ME
通讯作者：
Harris, ME
影响因子：
2.9
作者：
Hengge, AC;Zhao, Y;Zhang, ZY
通讯作者：
Zhang, ZY
影响因子：
15
作者：
HENGGE, AC;TOBIN, AE;CLELAND, WW
通讯作者：
CLELAND, WW
影响因子：
15
作者：
Hoff, RH;Mertz, P;Hengge, AC
通讯作者：
Hengge, AC
DOI：
10.1039/j29700001165
发表时间：
1970-01-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY B-PHYSICAL ORGANIC
影响因子：
--
作者：
KIRBY, AJ;YOUNAS, M
通讯作者：
YOUNAS, M