Lennette E P, Plapp B V
Biochemistry. 1979 Sep 4;18(18):3933-8. doi: 10.1021/bi00585a014.
The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.
研究了组氨酸乙内酰脲的咪唑基与溴乙酸酯的反应，以此作为蛋白质中组氨酸残基羧甲基化的模型。分别测定了咪唑环和乙内酰脲环的pK值为6.4和9.1（25℃），以及表观电离热为7.8和8.7千卡/摩尔。在对应于组氨酸乙内酰脲等电点的pH值下，测定了12、25、37和50℃时的羧甲基化速率；将修饰后的乙内酰脲水解为相应的组氨酸衍生物进行定量氨基酸分析。在pH 7.72和25℃下，咪唑的远-N烷基化（k = 3.9×10⁻⁵ M⁻¹ s⁻¹）的速度是近-N的两倍。单羧甲基衍生物在近-N处的羧甲基化速率相同（k = 2.1×10⁻⁵ M⁻¹ s⁻¹），但在远-N处快3倍（k = 11×10⁻⁵ M⁻¹ s⁻¹）。测定的咪唑环及其单羧甲基衍生物羧甲基化的活化焓相似（15.9±0.7千卡/摩尔）。四次羧甲基化的ΔS为-25±2 eu。根据25℃下介电常数对反应速率的影响计算出ΔS的静电分量（ΔS es）。单羧甲基化或二羧甲基化的ΔS es略为负值（-4±1 eu），表明过渡态中有一些电荷分离。所有四次羧甲基化的非静电活化熵为-21±2 eu。
