Regulatory Effect of Thiamin Pyrophosphate on Pig Heart Pyruvate Dehydrogenase Complex

Abstract

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 theKmvalue for pyruvate (from 76.7 to 19.0 μM) but a more moderate decrease ofKmfor CoA (from 12.2 to 4.3 μM) andKmfor NAD+(from 70.2 to 33.6 μM), with no considerable change in the maximum reaction rate. Likewise, thiamin monophosphate (TMP) decreased theKmvalue of PDC for pyruvate, but to a lesser extent (from 76.7 to 57.9 μM) than TPP. At the unsaturating level of pyruvate, theA50values for TPP and TMP were 0.2 μM 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.