Regulatory Properties of the Pyruvate-Dehydrogenase Complex from Escherichia coli. Thiamine Pyrophosphate as an Effector

Abstract

The pyruvate dehydrogenase complex from Escherichia coli is subject to an allosteric control. Pyruvate shows a homotropic cooperative effect. The extent of the cooperativity increases with decreasing concentrations of thiamine pyrophosphate. This cofactor itself exhibits positive cooperativity in steady-state measurements. The saturation curves both of pyruvate and of thiamine pyrophosphate have a very unusual asymmetric shape. The sigmoidal range is limited to low ligand concentrations and changes to a hyperbolic form far below half saturation. It was demonstrated that allosteric systems with more than eight interacting protomers, which obeys the allosteric model of Monod, J., Wyman, J., and Changeux, J.-P. [J. Mol. Biol. 12 (1965) 88-1171, behave in this manner.In the absence of substrate and thiamine pyrophosphate, the pyruvate dehydrogenase exists in an inactive form. At low thiamine pyrophosphate concentrations addition of pyruvate leads, after a lag phase of several minutes, to a partial activation. Within less than one second, complete activation occurs when saturating amounts of thiamine pyrophosphate are added, irrespective of the presence or absence of pyruvate. The lag period increases with decreasing degree of enzyme dilution. This phenomenon is not due to a dissociation and reassociation of subunits.Oxidative decarboxylation of pyruvate and transfer of the acetyl moiety formed to coenzyme A, corresponding to the overall reaction Pyruvate + NAD' + CoA Dihydrolipoyl-E2 + FAD-E3Reduced FAD-E3 + NAD'where El is pyruvate dehydrogenase, E2 dihydrolipoamide transacetylase, E3 dihydrolipoamide dehydrogenase and TPP, thiamine pyrophosphate. In the complex 16 identical subunits of each enzyme component are arranged to build up the native structure [3].The pyruvate dehydrogenase complex occupies a key position in metabolism as a link between glycolysis and the tricarboxylic acid cycle. Consequently the activity of this enzyme complex is influenced by several glycolytic intermediates [4 -61. Furthermore, the complex is subject to allosteric control, where pyruvate shows a positive cooperative effect, while acetyl-CoA is a strong allosteric inhibitor [7]. The target for nearly all effectors including acetyl-CoA is the pyruvate dehydrogenase component, which exhibits the cooperative effect of pyruvate also in the isolated, presumably Eur. J. Biochem. 48 (1974)

Dihydrolipoamide Dehydrogenase Mutation Alters the NADH Sensitivity of Pyruvate Dehydrogenase Complex of Escherichia coli K-12

“…In order to confirm that the lower sensitivity of the LPD from strain SE2378 to NADH inhibition is translated to the entire PDH complex, the PDH complex was purified from the wild type and two mutants, strains SE2377 and SE2378, representing the two alleles. Kinetic properties of the PDH from these strains are presented in (4,36). However, the native enzyme had a higher V max than the two mutated forms of the enzyme.…”

The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii

“…There is no kinetic evidence for such a site [ 2 ] . It is of interest to mention that Bisswanger [22] has shown that in the E. coli complex a small amount of TPP is very tightly bound to the complex. For reasons outlined below the low affinity binding site, i.e.…”

Multi-enzyme complexes in thermophilic organisms: isolation and characterization of the pyruvate dehydrogenase complex from Thermus aquaticus AT62

“…This may result from interactions of the non-polar hydrocarbon chains of the 2-oxoacids with the enzyme surface, which is more hydrophobic in thermophilic proteins than in mesophilic ones (Jaenicke, 1981 ;Zuber, 1981). Together with thiamin-diphosphate-dependent enzymes from other sources (Morey & Juni, 1968;Bisswanger, 1974;Butler et al, 1977) the T. aquaticus pyruvate dehydrogenase complex shares the poorly understood feature, that this cofactor, though being non-covalently bound, cannot be completely removed from the enzyme without destroying its catalytic activity.…”