Characterization of testis-specific isoenzyme of 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 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.
Korotchkina
Lioubov G
LG
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214, USA.
Sidhu
Sukhdeep
S
Patel
Mulchand S
MS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins; D013094:Spermatozoa / Q000502:physiology; D013737:Testis / Q000201:enzymology*
D007527:Isoenzymes; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D005947:Glucose