Strumilo S, Czerniecki J, Dobrzyn P
Department of Biochemistry, Institute of Biology, University of Bialystok, Bialystok, 15950, Poland.
Biochem Biophys Res Commun. 1999 Mar 16;256(2):341-5. doi: 10.1006/bbrc.1999.0321.
The kinetic behavior of pig heart pyruvate dehydrogenase complex (PDC) containing bound endogenous thiamin pyrophosphate (TPP) was affected by exogenous TPP. In the absence of exogenous TPP, a lag phase of the PDC reaction was observed. TPP added to the PDC reaction medium containing Mg2+ led to a disappearance of the lag phase, inducing strong reduction of the Km value for pyruvate (from 76.7 to 19.0 microM) but a more moderate decrease of Km for CoA (from 12.2 to 4.3 microM) and Km for NAD+ (from 70.2 to 33.6 microM), with no considerable change in the maximum reaction rate. Likewise, thiamin monophosphate (TMP) decreased the Km value of PDC for pyruvate, but to a lesser extent (from 76.7 to 57.9 microM) than TPP. At the unsaturating level of pyruvate, the A50 values for TPP and TMP were 0.2 microM and 0.3 mM, respectively. This could mean that the effect of TPP on PDC was more specific. In addition, exogenous TPP changed the UV spectrum and lowered the fluorescence emission of the PDC containing bound endogenous TPP in its active sites. The data obtained suggest that TPP plays, in addition to its catalytic function, the important role of positive regulatory effector of pig heart PDC.
含有内源性硫胺素焦磷酸（TPP）的猪心脏丙酮酸脱氢酶复合体（PDC）的动力学行为受到外源性TPP的影响。在没有外源性TPP的情况下，观察到PDC反应存在一个延迟期。向含有Mg2+的PDC反应介质中添加TPP会导致延迟期消失，丙酮酸的Km值大幅降低（从76.7微摩尔降至19.0微摩尔），但辅酶A的Km值降低幅度较小（从12.2微摩尔降至4.3微摩尔），NAD+的Km值也有所降低（从70.2微摩尔降至33.6微摩尔），最大反应速率没有显著变化。同样，硫胺素单磷酸（TMP）降低了PDC对丙酮酸的Km值，但降低程度比TPP小（从76.7微摩尔降至57.9微摩尔）。在丙酮酸不饱和水平下，TPP和TMP的A50值分别为0.2微摩尔和0.3毫摩尔。这可能意味着TPP对PDC的作用更具特异性。此外，外源性TPP改变了紫外光谱，并降低了活性位点含有内源性TPP的PDC的荧光发射。所获得的数据表明，TPP除了具有催化功能外，还对猪心脏PDC起着正调节效应器的重要作用。
