Characterization of testis‐specific human pyruvate dehydrogenase

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 α2β2 PDHs. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrate via pyruvate oxidation. Kinetic and regulatory properties of human PDH2 and human PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by substituting serines with alanine or glutamate in PDHs were investigated using four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2). PDH2 was found to be very similar to PDH1 in: (i) specific activities and kinetic parameters as determined by two functional assays; (ii); thermostability at 37° C; (iii) mechanism of inactivation by phosphorylation of three sites and (iv) phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated by: (i) 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) possible involvement of S264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) lower activities of PDK1, PDK2, PDK4 as well as PDP1 and PDP2 towards PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acid residues in PDH2. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure and hence preserving its kinetic and regulatory characteristics largely similar to that of PDH1. (Supported by NIH Grant DK20478.)