DECREASING THE BASICITY OF THE ACTIVE-SITE BASE, LYS-258, OF ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE BY REPLACEMENT WITH GAMMA-THIALYSINE
DECREASING THE BASICITY OF THE ACTIVE-SITE BASE, LYS-258, OF ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE BY REPLACEMENT WITH GAMMA-THIALYSINE
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DOI：
10.1021/bi00012a017
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发表时间：
1995-03-28
期刊：
影响因子：
2.9
通讯作者：
KIRSCH, JF
中科院分区：
文献类型：
--
作者：
GLOSS, LM;KIRSCH, JF
野生型天冬氨酸氨基转移酶(AATase)的K258C突变体与溴乙胺的烷基化反应与5种天然半胱氨酸的部分反应是复杂的[Planas，A.和Kirsch，J.F.(1991)生化30,8268-8276]。现在通过与K258C(Q)进行烷基化来克服这个问题，其中Cys-258是Quint中唯一的半胱氨酸残基，Quint是一种工程AATase，其中五个半胱氨酸已被转化为丙氨酸[Goss，L.M.，et al.(1992)生物化学31，32-39]。考察了K258C(Q)-EA的动力学和光谱性质，并与WT和Quint的结果进行了比较。用γ-硫代-赖氨酸取代Lys-258，导致内醛胺的pK(A)发生了1.3个pH单位的酸移。在D(k(Cat)/K-M(Asp))和D-kcat上，Quint的C-α氢动力学同位素效应分别为2.1和1.5。用较弱的碱取代Lys-258，使K258C(Q)-EA的配基亲和力和酮酸半反应的值分别增加到3.3和2.6，但氨基酸的k(CAT)/K-M值降低了一个数量级。K258C(Q)-EA和Quint的PMP的K-D值相等(0.2 nm)，比WT的K-D值低7倍。这些综合效应在L-天冬氨酸与K258C(Q)-EA与WT(和Quint)反应的自由能图中得到了说明。E。PLP和E.Quint的PMP络合物比WT的PMP络合物分别稳定0.9kcal/mol和1.1kcal/mol。E。K258C(Q)-EA的PLP形式比Quint稳定1.4kcal/mol，而相应的E。PMP络合物同样稳定。
Alkylation of the K258C mutant of the wild-type aspartate aminotransferase (AATase) with bromoethylamine to give gamma-thialysine 258 was complicated by partial reaction with the five native cysteines [Planas, A., and Kirsch, J. F. (1991) Biochemistry 30, 8268-8276]. This problem is now overcome by carrying out the alkylation with K258C(Q), in which Cys-258 is a unique cysteine residue in Quint, an engineered AATase in which the five cysteines have been converted to alanine [Gloss, L. M., et al. (1992) Biochemistry 31, 32-39]. The kinetics and spectral properties of the resulting enzyme, K258C(Q)-EA, have been examined and compared to those of WT and Quint. The replacement of Lys-258 by gamma-thia-Lys results in an acidic shift of 1.3 pH units in the pK(a) of the internal aldimine. The C-alpha hydrogen kinetic isotope effects for Quint are 2.1 and 1.5 on D(k(cat)/K-M(Asp)) and D-kcat, respectively. Replacement of Lys-258 by the weaker base, gamma-thia-Lys, increases these values to 3.3 and 2.6, respectively The changes of K258C(Q)-EA in ligand affinities and the keto acid half-reaction are minor; however, the k(cat)/K-M values for amino acids are decreased by an order of magnitude. The K-D values for PMP of K258C(Q)-EA and Quint are equal to each other (0.2 nM) and are 7-fold lower than that of WT. These combined effects are illustrated in the free energy diagrams of the reaction with L-Asp with K258C(Q)-EA, relative to WT (and Quint). The E . PLP and E . PMP complexes of Quint are 0.9 and 1.1 kcal/mol, respectively, more stable than those of WT. The E . PLP form of K258C(Q)-EA is 1.4 kcal/mol more stable than that of Quint, while the corresponding E . PMP complexes are equally stable.