Abstract
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 alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. 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. 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 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. 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. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. 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.
Highlights
Lation of the lipoyl moieties of dihydrolipoamide acetyltransferase (E2); (ii) E2 transferring acetyl moiety to CoA; (iii) and dihydrolipoamide dehydrogenase (E3) reoxidizing the reduced lipoyl moieties of E2 with the reduction of NADϩ to NADH [1,2,3]
In this study we have examined the catalytic parameters of human PDH2 and compared them with that of PDH1, and we investigated the mechanism of inactivation by phosphorylation of each of the three sites of PDH2 and the specificity of the four pyruvate dehydrogenase kinase (PDK) isoenzymes and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes for the three phosphorylation sites of PDH2
The PDH2 and PDH1 isoenzymes have highly conserved sequences in the TPP motif region (sequence of residues conserved within the TPP-requiring enzymes, residues of which are involved in the binding of the pyrophosphate moiety of TPP through the divalent ion [28, 29])
Summary
Materials—All chemicals were of high purity and were obtained from Sigma. Restriction and DNA-modifying enzymes were purchased from Promega (Madison, WI). The final construct (pET-28b-PDHA2/PDHB) contained the PDHA2 cDNA and PDHB cDNA His tag was added to the N terminus of the PDH2 sequence for affinity purification with Ni-NTA-agarose column. L2S was immobilized on the CM5 chip through the lipoyl moieties by a surface thiol coupling method For this purpose the following reagents were applied on the sensor chip surface at a flow rate of 5 l/min: 35 l of the 1:1 mixture of 100 mM N-hydroxysuccinimide and 100 mM N-ethyl-NЈ-[3(dimethylamino)propyl]carbodiimide hydrochloride to activate the surface; 15 l of 40 mM cystamine in 100 mM sodium borate buffer, pH 8.5, to introduce disulfides; 20 l of 100 mM dithioerythritol in 100 mM. Determination of PDKs Activity and Kinetic Parameters—Activities of PDKs were determined with double mutants of PDH2 and PDH1 (having only site 1–3 available for phosphorylation) either after reconstitution in PDC or in a free form as described previously [13].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
