Pas H H, ten Hoeve-Duurkens R H, Robillard G T
Department of Physical Chemistry, University of Groningen, The Netherlands.
Biochemistry. 1988 Jul 26;27(15):5520-5. doi: 10.1021/bi00415a020.
The amino acid composition and sequence of EIIMtl is known [Lee, C. A., & Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 10761-10767]. This information was combined, in the present study, with quantitative amino acid analysis to determine the molar concentration of the enzyme. The stoichiometry of phosphoryl group incorporation was then determined by phosphorylation of enzyme II from [14C]-phosphoenolpyruvate (pyruvate burst procedure). The native, reduced enzyme incorporated two phosphoryl groups per monomer. Both phosphoryl groups were shown to be transferred to mannitol. Oxidation or N-ethylmaleimide (NEM) labeling of Cys-384 resulted in incorporation of only one phosphoryl group per monomer, which was unable to be transferred to mannitol. The number of mannitol binding sites on enzyme II was determined by centrifugation using Amicon Centricon microconcentrators. The reduced unphosphorylated enzyme contained one high-affinity binding site (KD = 0.1 microM) per dimer and a second site with a KD in the micromolar range. Oxidation or NEM labeling did not change the number of binding sites.
EIIMtl的氨基酸组成和序列已为人所知[Lee, C. A., & Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 10761 - 10767]。在本研究中，将该信息与定量氨基酸分析相结合，以确定该酶的摩尔浓度。然后通过[14C]-磷酸烯醇丙酮酸对酶II进行磷酸化（丙酮酸爆发程序）来确定磷酰基掺入的化学计量。天然的、还原态的酶每个单体掺入两个磷酰基。两个磷酰基均显示被转移至甘露醇。对Cys-384进行氧化或N-乙基马来酰亚胺（NEM）标记后，每个单体仅掺入一个磷酰基，且该磷酰基无法转移至甘露醇。使用Amicon Centricon微浓缩器通过离心法确定酶II上甘露醇结合位点的数量。还原态的未磷酸化酶每个二聚体含有一个高亲和力结合位点（KD = 0.1 microM）和另一个KD在微摩尔范围内的位点。氧化或NEM标记并未改变结合位点的数量。
