Wrobel J A, Stinson R A
Eur J Biochem. 1978 Apr 17;85(2):345-50. doi: 10.1111/j.1432-1033.1978.tb12245.x.
The single thiol of yeast phosphoglycerate kinase was labelled with the chromophoric sulfhydryl reagent, 2-chloromercuri-4-nitrophenol. Sequential additions of individual anions to this modified enzyme brought about a decrease in absorbance at 410 nm that reflected the degree of saturation of the enzyme with anion. The binding curves were analyzed to determine the dissociation constants of a number of anions with charges varying from--1 to--4.1. A linear relationship was found between the charge of the anion and the negative logarithm of the dissociation constant for the labelled enzyme-anion complex. The highly charged anions, such as ATP, bound more tightly than did anions with less charge, such as Cl-. The average number of binding sites for those anions for which accurate results could be obtained was 1.06 mol per 47000 g of enzyme. Several lines of evidence suggested that titration of the active center was not being monitored. Anions bound to phosphoglycerate kinase decreased the rate of reaction between the enzyme thiol and 5,5'-dithiobis(2-nitrobenzoic acid). The relationship between the degree of saturation of the anion binding site and the reaction rate constant was used to calculate the dissociation constant between anion and enzyme. Dissociation constants determined in this manner were in good agreement with those determined by titration of the enzyme-mercurial complex.
酵母磷酸甘油酸激酶的单一巯基用发色巯基试剂2-氯汞基-4-硝基苯酚进行标记。向这种修饰后的酶中依次添加各种阴离子，导致410nm处吸光度下降，这反映了酶与阴离子的饱和程度。对结合曲线进行分析，以确定多种电荷从-1到-4.1不等的阴离子的解离常数。发现阴离子电荷与标记的酶-阴离子复合物解离常数的负对数之间存在线性关系。高电荷阴离子，如ATP，比低电荷阴离子，如Cl-，结合更紧密。对于能够获得准确结果的那些阴离子，其平均结合位点数为每47000g酶1.06mol。几条证据表明，监测的不是活性中心的滴定。与磷酸甘油酸激酶结合的阴离子降低了酶巯基与5,5'-二硫代双(2-硝基苯甲酸)之间的反应速率。利用阴离子结合位点的饱和程度与反应速率常数之间的关系来计算阴离子与酶之间的解离常数。以这种方式确定的解离常数与通过滴定酶-汞复合物确定的解离常数高度一致。
