Woolfitt A R, Kellett G L, Hoggett J G
Department of Biology, University of York, U.K.
Biochim Biophys Acta. 1988 Jan 29;952(2):238-43. doi: 10.1016/0167-4838(88)90121-5.
The binding of glucose, ADP and AdoPP[NH]P, to the native PII dimer and PII monomer and the proteolytically-modified SII monomer of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from Saccharomyces cerevisiae was monitored at pH 6.7 by the concomitant quenching of protein fluorescence. The data were analysed in terms of Qmax, the maximal quenching of fluorescence at saturating concentrations of ligand, and [L]0.5, the concentration of ligand at half-maximal quenching. No changes in fluorescence were observed with free enzyme and nucleotide alone. In the presence of saturating levels of glucose, Qmax induced by nucleotide was between 2 and 7%, and [L]0.5 was between 0.12 and 0.56 mM, depending on the nucleotide and enzyme species. Qmax induced by glucose alone was between 22 and 25%, while [L]0.5 was approx. 0.4 mM for either of the monomeric hexokinase forms and 3.4 for PII dimer. In the presence of 6 mM ADP or 2 mM AdoPP[NH]P, Qmax for glucose was increased by up to 4% and [L]0.5 was diminished 3-fold for hexokinase PII monomer, 6-fold for SII monomer, and 15-fold for PII dimer. The results are interpreted in terms of nucleotide-induced conformational change of hexokinase in the presence of glucose and synergistic binding interactions between glucose and nucleotide.
在pH 6.7条件下，通过蛋白质荧光的伴随猝灭监测葡萄糖、ADP和AdoPP[NH]P与酿酒酵母己糖激酶（ATP：D-己糖6-磷酸转移酶，EC 2.7.1.1）的天然PII二聚体、PII单体以及经蛋白水解修饰的SII单体的结合情况。根据Qmax（配体饱和浓度下荧光的最大猝灭）和[L]0.5（荧光猝灭一半时的配体浓度）对数据进行分析。单独的游离酶和核苷酸未观察到荧光变化。在葡萄糖饱和水平存在时，核苷酸诱导的Qmax在2%至7%之间，[L]0.5在0.12至0.56 mM之间，具体取决于核苷酸和酶的种类。单独葡萄糖诱导的Qmax在22%至25%之间，而[L]0.5对于任何一种单体己糖激酶形式约为0.4 mM，对于PII二聚体为3.4 mM。在存在6 mM ADP或2 mM AdoPP[NH]P时，己糖激酶PII单体的葡萄糖Qmax增加高达4%，[L]0.5降低3倍；SII单体降低6倍；PII二聚体降低15倍。结果从葡萄糖存在下核苷酸诱导的己糖激酶构象变化以及葡萄糖与核苷酸之间的协同结合相互作用方面进行了解释。
