![]() ![]() PDK2 had the highest activity for site 1 PDK3 had higher activity for site 2 than for site 1, and only PDK1 could phosphorylate site 3. PDKs had site specificity toward the three phosphorylation sites of PDH2 similar to that toward the three phosphorylation sites of PDH1. ![]() Double mutants of phosphorylation sites of PDH2 and PDH1, having only site 1 (PDH-S2A/S3A), site 2 (PDH-S1A/S3A), or site 3 (PDH-S1A/S2A) were used for these experiments. ![]() 4 shows activities of PDK isoenzymes for the three phosphorylation sites of PDH2 and PDH1 when both PDH and PDK were reconstituted in PDC. In this study we have investigated if PDK isoenzymes phosphorylate the three phosphorylation sites of PDH2 differently than those of PDH1. The results indicate that the affinity of PDH2 for the PDH-binding domain of E2 differs only modestly from that of PDH1 however, about 2-fold faster association of PDH2 with L2S results in the lower KD value. The rate constants of association were 9.9 × 10 5 and 4.0 × 10 5 m -1 s -1 for PDH2 and PDH1, respectively the rate constants of dissociation were 0.0172 and 0.0170 s -1 for PDH2 and PDH1, respectively and the equilibrium dissociation constants were 17.5 and 42.5 n m for PDH2 and PDH1, respectively. 3 shows the sensorgrams obtained with different concentrations of PDH2 and PDH1. By using this immobilization approach, L2S was bound to the chip through the lipoyl domain leaving the PDH-binding domain available for PDH binding and not affected by immobilization because the two domains of L2S are connected by a flexible hinge region. L2S consisting of the second lipoyl domain, second hinge region, PDH-binding domain, and the third hinge region was immobilized on the CM5 chip by a surface thiol coupling method. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1.īinding of PDH2 to the PDH-binding Domain of E2-Binding of PDH2 and PDH1 with the PDH-binding domain of E2 was investigated by surface plasmon resonance. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 α subunit. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay (ii) in thermostability at 37 ☌ (iii) in the mechanism of inactivation by phosphorylation of three sites and (iv) in the phosphorylation of sites 1 and 2 by PDK3. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. ![]() Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the α subunit of α 2 β 2 PDH.
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