TAILIEUCHUNG - Báo cáo khóa học: C-, 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins

Thioredoxin reductase (TrxR) fromEscherichia coli,the mutant proteins E159Y and C138S, and the mutant protein C138Streated with phenylmercuric acetate were reconstituted with [U-13 C17 ,U-15 N4]FAD and analysed, in their oxidized and reduced states, by 13 C-, 15 N- and 31 P-NMR spectroscopy. The enzymes studied showed very similar 31 P-NMR spectra in the oxidized state, con-sisting of two peaks at ) and ) . In the reduced state, the two peaks merge into one apparent peak (at) .) | Eur. J. Biochem. 271 1437-1452 2004 FEBS 2004 doi 13C- 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins Wolfgang Eisenreich1 Kristina Kemter1 Adelbert Bacher1 Scott B. Mulrooney2 Charles H. Williams Jr2 3 and Franz Muller4 1Lehrstuhl fur Organische Chemie und Biochemie Technische Universitat MUnchen Germany .Department of Biological Chemistry The University of Michigan Ann Arbor Michigan USA 3Department of Veterans Affairs Medical Center Ann Arbor Michigan USA 4Wylstrasse 13 Hergiswil Switzerland Thioredoxin reductase TrxR from Escherichia coli the mutant proteins E159Y and C138S and the mutant protein C138S treated with phenylmriuucc ccetate wrre reconstituted with U-13C-7 U-15N4 FAD and analysed in their oxidized and reduced states by -3C- -5N- and 31P-NMR spectroscopy. The enzymes studied showed very similar 31P-NMR spectra in the oxidized state consisting of two peaks at and . In the reduced state the two peaks merge into one apparent peak at . . The data are compared with published 31P-NMR data of enzymes closely related to TrxR. 13C and 15N-NMR chemical shifts of TrxR and the mutant proteins in the oxidized state provided information about the electronic structure of the protein-bound cofactor and its interactions with the apoproteins. Strong hydrogen bonds exist between protein-bound flavin and the apoproteins at C 2 O C 4 O N 1 and N 5 . The N 10 atoms in the enzymes are slightly out of the molecular plane of the flavin. Of the ribityl carbon atoms C 10a Y S are the most affected upon binding to the apoprotein and the large downfield shift of the C 10y atom indicates strong hydrogen bonding with the apoprotein. The hydrogen bonding pattern observed is in excellent agreement with X-ray data except for the N 1 and the N 3 atoms where a reversed situation was observed. Some chemical shifts observed in C138S deviate considerably from .

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