Allosteric coupling in pyruvate dehydrogenase kinase 2.
Mitochondrial pyruvate dehydrogenase kinase 2 (PDHK2) phosphorylates the pyruvate dehydrogenase multienzyme complex (PDC) and thereby controls the rate of oxidative decarboxylation of pyruvate. The activity of PDHK2 is regulated by a variety of metabolites such as pyruvate, NAD (+), NADH, CoA, and acetyl-CoA. The inhibitory effect of pyruvate occurs through the unique binding site, which is specific for pyruvate and its synthetic analogue dichloroacetate (DCA). The effects of NAD (+), NADH, CoA, and acetyl-CoA are mediated by the binding site that recognizes the inner lipoyl-bearing domain (L2) of the dihydrolipoyl transacetylase (E2). Both allosteric sites are separated from the active site of PDHK2 by more than 20 A. Here we show that mutations of three amino acid residues located in the vicinity of the active site of PDHK2 (R250, T302, and Y320) make the kinase resistant to the inhibitory effect of DCA, thereby uncoupling the active site from the allosteric site. In addition, we provide evidence that substitutions of R250 and T302 can partially or completely uncouple the L2-binding site. Based on the available structural data, R250, T302, and Y320 stabilize the "open" and "closed" conformations of the built-in lid that controls the access of a nucleotide into the nucleotide-binding cavity. This strongly suggests that the mobility of ATP lid is central to the allosteric regulation of PDHK2 activity serving as a conformational switch required for communication between the active site and allosteric sites in the kinase molecule.
Klyuyeva
Alla
A
Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Tuganova
Alina
A
Popov
Kirill M
KM
D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site / Q000235:genetics; D000818:Animals; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D051381:Rats
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D017346:Protein Serine-Threonine Kinases