| Autor: |
Shisler KA; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Hutcheson RU; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Horitani M; Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States., Duschene KS; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Crain AV; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Byer AS; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Shepard EM; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Rasmussen A; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Yang J; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Broderick WE; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States., Vey JL; Department of Chemistry and Biochemistry, California State University Northridge , Northridge, California 91330, United States.; Departments of Chemistry and Biology and the Howard Hughes Medical Institute, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States., Drennan CL; Departments of Chemistry and Biology and the Howard Hughes Medical Institute, Massachusetts Institute of Technology , Cambridge, Massachusetts 02139, United States., Hoffman BM; Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States., Broderick JB; Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States. |
| Abstrakt: |
Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster. |
