Heterogeneity of binding sites for the pyruvate dehydrogenase component on the dihydrolipoyl transacetylase core of bovine kidney pyruvate dehydrogenase complex.
We have characterized the dissociation equilibrium constant (Kd) and the rate constants of association and dissociation for the binding of the pyruvate dehydrogenase component (PDH) to the dihydrolipoyl transacetylase component of kidney pyruvate dehydrogenase complex. We have found about 7 high-affinity sites (Kd = 1.5 X 10(-11) M) and about 13 low-affinity sites (Kd = 2.5 X 10(-8) M) with negative cooperativity for binding of PDH at the weaker sites. The high-affinity sites show a much higher rate constant for association of PDH with the core than do the low-affinity sites. Catalytic turnover strengthens binding of PDH at high-affinity sites when the complex is preincubated in the presence of thiamin pyrophosphate (TPP). In the absence of TPP, appreciably tighter binding occurs at the low-affinity sites (Kd = 5.9 X 10(-10) M). TPP weakens PDH binding at these sites with a half-maximal effect at about 5 microM TPP. In addition to TPP, increased ionic strength or 1-3 mM Mg2+ (with an enhanced effect of Mg2+ at higher ionic strengths) weakens PDH binding at low-affinity sites with a corresponding increase in the rate constant for dissociation. Further studies will be required to determine whether site heterogeneity contributes to dynamic processes in the function and regulation of the pyruvate dehydrogenase complex.
D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D005033:Ethylmaleimide / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008433:Mathematics; D009994:Osmolar Concentration; D011485:Protein Binding; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*
D011768:Pyruvate Dehydrogenase Complex; D015636:Magnesium Chloride; D030481:Pyruvate Dehydrogenase (Lipoamide); D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D008274:Magnesium; D005033:Ethylmaleimide