Han M K, Roseman S, Brand L
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
J Biol Chem. 1990 Feb 5;265(4):1985-95.
Enzyme I is the first protein of the phospho transfer sequence in the bacterial phosphoenolpyruvate:glycose phosphotransferase system. This protein exhibits a temperature-dependent monomer/dimer equilibrium. The nucleotide sequence of Escherichia coli ptsI indicates four -SH residues per subunit (Saffen, D. W., Presper, K. A., Doering, T. L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253). In the present experiments, the sulfhydryl groups of the E. coli enzyme were studied with various -SH-specific reagents. Titration of Enzyme I with 5,5'-dithiobis-2-nitrobenzoic acid also revealed four reacting -SH groups. The kinetics of the 5,5'-dithiobis-2-nitrobenzoic acid reaction with Enzyme I exhibit biphasic character, with pseudo-first order rate constants of 2.3 x 10(-2)/s and 2.3 x 10(-3)/s at pH 7.5, at room temperature. Fractional amplitudes associated with the rate constants were 25 +/- 5% for the fast and 75 +/- 5% for the slow rate. The "slow" rate was influenced by ligands that react with Enzyme I (the protein HPr, Mg2+, Mg2+ plus P-enolpyruvate), and also by temperature (at the temperature range where the monomer/dimer association occurs). The fractional ratio of the two rates remained at 1:3 under these conditions. Thus, under all conditions tested, two classes of -SH groups were detected, one reacting more rapidly than the other three -SH groups. Modification of the "fast" -SH group results in an active enzyme capable of forming dimer, whereas modification of the slow -SH groups results in inactive and monomeric Enzyme I. The enzyme was labeled with pyrene maleimide under conditions where only the more reactive sulfhydryl group was derivatized. Hydrolysis by trypsin followed by reverse-phase high performance liquid chromatography analysis of the peptide mixture resulted in only one fluorescent peak. This peak was not observed when the more reactive sulfhydryl residue was protected prior to pyrene maleimide labeling. Amino acid sequencing of the fluorescent peak indicated that the more reactive residue is the C-terminal amino acid residue, cysteine 575. The results provide a means for selectively labeling Enzyme I with a fluorophore at a single site while retaining full catalytic activity.
酶I是细菌磷酸烯醇丙酮酸：葡萄糖磷酸转移酶系统中磷酸转移序列的首个蛋白质。该蛋白质呈现出温度依赖性的单体/二聚体平衡。大肠杆菌ptsI的核苷酸序列表明每个亚基有四个-SH残基（萨芬，D.W.，普雷斯珀，K.A.，多林，T.L.，和罗斯曼，S.（1987年）《生物化学杂志》262，16241 - 16253）。在本实验中，用各种-SH特异性试剂研究了大肠杆菌酶的巯基。用5,5'-二硫代双-2-硝基苯甲酸滴定酶I也揭示了四个反应性-SH基团。5,5'-二硫代双-2-硝基苯甲酸与酶I反应的动力学呈现双相特征，在pH 7.5、室温下，假一级速率常数分别为2.3×10⁻²/s和2.3×10⁻³/s。与速率常数相关的分数幅度，快速反应的为25±5%，慢速反应的为75±5%。“慢速”反应速率受与酶I反应的配体（蛋白质HPr、Mg²⁺、Mg²⁺加磷酸烯醇丙酮酸）影响，也受温度影响（在单体/二聚体缔合发生的温度范围内）。在这些条件下，两种速率的分数比保持在1:3。因此，在所有测试条件下，检测到两类-SH基团，一类比其他三个-SH基团反应更快。修饰“快速”的-SH基团会产生一种能够形成二聚体的活性酶，而修饰慢速的-SH基团会导致无活性的单体酶I。在仅使反应性更强的巯基被衍生化的条件下，用芘马来酰亚胺标记该酶。用胰蛋白酶水解，然后对肽混合物进行反相高效液相色谱分析，只得到一个荧光峰。当在芘马来酰亚胺标记之前保护反应性更强的巯基残基时，未观察到这个峰。对荧光峰进行氨基酸测序表明，反应性更强的残基是C末端氨基酸残基半胱氨酸575。这些结果提供了一种在保留完全催化活性的同时在单个位点用荧光团选择性标记酶I的方法。
