Neuzil J, Danielson H, Welch G R, Ovádi J
Institute of Enzymology, Hungarian Academy of Sciences, Budapest.
Biochim Biophys Acta. 1990 Mar 1;1037(3):307-12. doi: 10.1016/0167-4838(90)90030-j.
The combination of binding and kinetic approaches is suggested to study (i) the mechanism of substrate-modulated dynamic enzyme associations; (ii) the specificity of enzyme interactions. The effect of complex formation between aldolase and glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) on aldolase catalysis was investigated under pseudo-first-order conditions. No change in kcat but a significant increase in KM of fructose 1,6-bisphosphate for aldolase was found when both enzymes were obtained from muscle. In contrast, kcat rather than KM changed if dehydrogenase was isolated from yeast. Next, the conversion of fructose 1-phosphate was not affected by interactions between enzyme couples isolated from muscle. The influence of fructose phosphates on the enzyme-complex formation was studied by means of covalently attached fluorescent probe. We found that the interaction ws not perturbed by the presence of fructose 1-phosphate; however, fructose 1,6-bisphosphate altered the dissociation constant of the enzyme complex. A molecular model for fructose 1,6-bisphosphate-modulated enzyme interaction has been evaluated which suggests that high levels of fructose bisphosphate would drive the formation of the 'channelling' complex between aldolase and glyceraldehyde-3-phosphate dehydrogenase.
（i）底物调节的动态酶缔合机制；（ii）酶相互作用的特异性。在准一级条件下，研究了醛缩酶与3-磷酸甘油醛脱氢酶（D-甘油醛-3-磷酸：NAD⁺氧化还原酶（磷酸化），EC 1.2.1.12）之间形成复合物对醛缩酶催化作用的影响。当两种酶均从肌肉中获得时，发现醛缩酶的kcat没有变化，但1,6-二磷酸果糖的KM显著增加。相反，如果脱氢酶是从酵母中分离出来的，则kcat而不是KM发生了变化。接下来，1-磷酸果糖的转化不受从肌肉中分离出的酶对之间相互作用的影响。通过共价连接的荧光探针研究了果糖磷酸酯对酶复合物形成的影响。我们发现，1-磷酸果糖的存在不会干扰这种相互作用；然而，1,6-二磷酸果糖改变了酶复合物的解离常数。已经评估了1,6-二磷酸果糖调节的酶相互作用的分子模型，该模型表明高浓度的果糖二磷酸酯会促使醛缩酶与3-磷酸甘油醛脱氢酶之间形成“通道化”复合物。
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