Tijane M N, Chaffotte A F, Seydoux F J, Roucous C, Laurent M
J Biol Chem. 1980 Nov 10;255(21):10188-93.
Yeast phosphofructokinase contains 83 +/- 2 cysteinyl residues/enzyme oligomer. On the basis of their reactivity toward 5,5-dithiobis(2-nitrobenzoic acid), the accessible cysteinyl residues of the native enzyme may be classified into three groups. For titrations performed with N-ethylmaleimide, subdivisional classes of reactivity are evidenced. In each case, the 6 to 8 most reactive cysteines are not protected by fructose 6-phosphate from chemical labeling and do not seem involved in subsequent enzyme inactivation. Differential labeling studies as well as direct protection experiments in the presence of fructose 6-phosphate, indicate that 12 -SH groups/enzyme oligomer (i.e. three -SH groups per binding site) are protected by the allosteric substrate from the chemical modification. Specific labeling by the differential method of the cysteinyl residues protected by fructose 6-phosphate and further separation of the two types of subunits constituting yeast phosphofructokinase, show that the substrate binding sites are localized exclusively on subunits of beta type. Thus, alpha subunits are not implicated directly in the catalytic mechanism of yeast phosphofructokinase reaction.
酵母磷酸果糖激酶每个酶寡聚体含有83±2个半胱氨酰残基。根据它们对5,5-二硫代双（2-硝基苯甲酸）的反应性，天然酶中可及的半胱氨酰残基可分为三组。在用N-乙基马来酰亚胺进行的滴定中，显示出反应性的细分类别。在每种情况下，6至8个反应性最强的半胱氨酸不受6-磷酸果糖的化学标记保护，似乎也不参与随后的酶失活。差异标记研究以及在6-磷酸果糖存在下的直接保护实验表明，别构底物可保护每个酶寡聚体中的12个巯基（即每个结合位点3个巯基）免受化学修饰。通过差异方法对受6-磷酸果糖保护的半胱氨酰残基进行特异性标记，并进一步分离构成酵母磷酸果糖激酶的两种亚基，结果表明底物结合位点仅位于β型亚基上。因此，α亚基不直接参与酵母磷酸果糖激酶反应的催化机制。
