Esakova O A, Khanova E A, Meshalkina L E, Golbik R, Hübner G, Kochetov G A
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
Biochemistry (Mosc). 2005 Jul;70(7):770-6. doi: 10.1007/s10541-005-0182-4.
The influence of transketolase substrates on the interaction of apotransketolase with its coenzyme thiamine diphosphate (TDP) and on the stability of the reconstituted holoenzyme was studied. Donor substrates increased the affinity of the coenzyme for transketolase, whereas acceptor substrate did not. In the presence of magnesium ions, the active centers of transketolase initially identical in TDP binding lose their equivalence in the presence of donor substrates. The stability of transketolase depended on the cation type used during its reconstitution--the holoenzyme reconstituted in the presence of calcium ions was more stable than the holoenzyme produced in the presence of magnesium ions. In the presence of donor substrate, the holoenzyme stability increased without depending on the cation used during the reconstitution. Donor substrate did not influence the interaction of apotransketolase with the inactive analog of the coenzyme N3'-pyridyl thiamine diphosphate and did not stabilize the transketolase complex with this analog. The findings suggest that the effect of the substrate on the interaction of the coenzyme with apotransketolase and on stability of the reconstituted holoenzyme is caused by generation of 2-(alpha,beta-dihydroxyethyl)thiamine diphosphate (an intermediate product of the transketolase reaction), which has higher affinity for apotransketolase than TDP.
研究了转酮醇酶底物对脱辅基转酮醇酶与其辅酶硫胺二磷酸（TDP）相互作用以及重组全酶稳定性的影响。供体底物增加了辅酶对转酮醇酶的亲和力，而受体底物则没有。在镁离子存在下，转酮醇酶的活性中心最初在TDP结合方面是相同的，但在供体底物存在时失去了等效性。转酮醇酶的稳定性取决于其重组过程中使用的阳离子类型——在钙离子存在下重组的全酶比在镁离子存在下产生的全酶更稳定。在供体底物存在下，全酶稳定性增加，且不依赖于重组过程中使用的阳离子。供体底物不影响脱辅基转酮醇酶与辅酶N3'-吡啶基硫胺二磷酸的无活性类似物的相互作用，也不能稳定转酮醇酶与该类似物的复合物。研究结果表明，底物对辅酶与脱辅基转酮醇酶相互作用以及重组全酶稳定性的影响是由2-(α,β-二羟乙基)硫胺二磷酸（转酮醇酶反应的中间产物）的生成引起的，该产物对脱辅基转酮醇酶的亲和力高于TDP。
