tailieunhanh - Báo cáo Y học: Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehy-drogenase complex (PDC).Analysis of the primary amino-acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases, despite the fact that PDKexclusivelyphosphorylates Ser residues in theE1a subunit of the seeming contradiction might be resolved if the PDK-catalyzed reaction employed a phos-pho-His intermediate | Eur. J. Biochem. 269 2601-2606 2002 FEBS 2002 doi Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase Alejandro Tovar-Mendez1 Jan A. Miernyk1 2 and Douglas D. Randall1 1 Department of Biochemistry University of Missouri Columbia MO 65211 USA 2USDA Agricultural Research Service Plant Genetics Research Unit Columbia MO 65211 USA Pyruvate dehydrogenase kinase PDK is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex PDC . Anialysis of the primary aminoacid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two-component His-kinases despite the fact that PDK exclusively phosphorylates Ser residues in the E1a subunit of the PDC. This seeming conl adiciion might be resolved if the PDK-catalyzed reaction employed a phos-pho-His intermediate. The nsuulte horn pHitlabilily cu cSsss of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho-His intermediate. Furthermore site-directed mutagenesis of the two most likely phosphotransfer His residues H121 and H168 did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1a. Thus PDK ÍS a Liniq Lie yype of protein kinase having a His-kinase-like sequence but Ser-kinase activity. Keywords autophosphorylation protein kinase pyruvate dehydrogenase complex regulatory phosphorylation site-directed mutagenesis. The reaction catalyzed by the pyruvate dehydrogenase complex PDC occupies a key position in intermediary metabolism and is subject to multiple layers of regulation 1 2 . Revessible phosphoryaiiíon ÍS a parAGu y important control mechanism for mitochondrial PDC 3 4 . Mulsisise serine phosphorylation of the E1a subunit of PDC by the intrinsic pyruvate dehydrogenase kinase PDK inactivates the complex which can then be re-activated by an intrinsic phosphopyruvate dehydrogenase phosphatase 5 . Se i --state activity of