Properties and subunit structure of pig heart pyruvate dehydrogenase.
Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D009905:Optical Rotatory Dispersion; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D013835:Thiamine Pyrophosphate / Q000494:pharmacology; D014357:Trypsin
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011768:Pyruvate Dehydrogenase Complex; D013439:Sulfhydryl Reagents; D014357:Trypsin; D008274:Magnesium; D013835:Thiamine Pyrophosphate; D002118:Calcium