Vas M, Batke J
Eur J Biochem. 1984 Feb 15;139(1):115-23. doi: 10.1111/j.1432-1033.1984.tb07984.x.
Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group.
为监测底物与猪肌肉3-磷酸甘油酸激酶的结合情况，将其与1-苯胺基-8-萘磺酸盐（ANS）复合。在所使用的实验条件下，酶与染料的相互作用不影响酶活性。通过测量紧密结合在酶上的ANS分子荧光发射中底物依赖性变化（解离常数Kd≤0.05 mM），于20℃在含5 mM巯基乙醇的0.1 M Tris/HCl缓冲液（pH 7.5）中进行荧光滴定。3-磷酸甘油酸、MgATP、1,3-二磷酸甘油酸和MgADP单独结合的解离常数分别为0.03±0.01 mM、0.15±0.10 mM、0.00005±0.00001 mM和0.15±0.10 mM。当MgATP也结合到酶上时，3-磷酸甘油酸的结合减弱：在此三元复合物中3-磷酸甘油酸的解离常数（0.25±0.08 mM）与其Km值（0.38±0.10 mM）相当。在非生产性三元复合物中，当MgATP被MgADP（Kd = 0.20±0.10 mM）或AMP（Kd = 0.12±0.05 mM）取代时，可观察到同样的减弱情况，而腺苷则无此作用。这表明核苷酸带负电荷的磷酸基团在影响3-磷酸甘油酸结合方面的重要性。与3-磷酸甘油酸相反，底物类似物甘油3-磷酸的结合实际上不受MgATP存在的影响：其与游离酶的解离常数（0.40±0.10 mM）与其抑制常数（0.70±0.20 mM）相当。这一发现以及甘油3-磷酸结合的解离常数（0.40±0.10 mM）与3-磷酸甘油酸的Km值（0.38±0.10 mM）的相似性表明,在酶促反应过程中，3-磷酸甘油酸的结合可能不涉及羧基。
