Phosphorylation of Serine 264 Impedes Active Site Accessibility in the E1 Component of the Human Pyruvate Dehydrogenase Multienzyme Complex

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• 作者：Franziska Seifert ; Ewa Ciszak ; Lioubov Korotchkina ; Ralph Golbik ; Michael Spinka ; Paulina Dominiak ; Sukhdeep Sidhu ; Johanna Brauer ; Mulchand S. Patel ; Kai Tittmann
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：May 29, 2007
• 年：2007
• 卷：46
• 期：21
• 页码：6277 - 6287
• 全文大小：1343K
• 年卷期：v.46,no.21(May 29, 2007)
• ISSN：1520-4995

文摘

At the junction of glycolysis and the Krebs cycle in cellular metabolism, the pyruvatedehydrogenase multienzyme complex (PDHc) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. In mammals, PDHc is tightly regulated by phosphorylation-dephosphorylation of three serine residuesin the thiamin-dependent pyruvate dehydrogenase (E1) component. In vivo, inactivation of human PDHccorrelates mostly with phosphorylation of serine 264, which is located at the entrance of the substratechannel leading to the active site of E1. Despite intense investigations, the molecular mechanism of thisinactivation has remained enigmatic. Here, a detailed analysis of microscopic steps of catalysis in humanwild-type PDHc-E1 and pseudophosphorylation variant Ser264Glu elucidates how phosphorylation ofSer264 affects catalysis. Whereas the intrinsic reactivity of the active site in catalysis of pyruvatedecarboxylation remains nearly unaltered, the preceding binding of substrate to the enzyme's active sitevia the substrate channel and the subsequent reductive acetylation of the E2 component are severelyslowed in the phosphorylation variant. The structure of pseudophosphorylation variant Ser264Gludetermined by X-ray crystallography reveals no differences in the three-dimensional architecture of thephosphorylation loop or of the active site, when compared to those of the wild-type enzyme. However,the channel leading to the active site is partially obstructed by the side chain of residue 264 in the variant.By analogy, a similar obstruction of the substrate channel can be anticipated to result from a phosphorylation of Ser264. The kinetic and thermodynamic results in conjunction with the structure of Ser264Glusuggest that phosphorylation blocks access to the active site by imposing a steric and electrostaticbarrier for substrate binding and active site coupling with the E2 component. As a Ser264Gln variant,which carries no charge at position 264, is also selectively deficient in pyruvate binding andreductive acetylation of E2, we conclude that mostly steric effects account for inhibition of PDHc byphosphorylation.