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By  and Anand Balakrishnan and Anand Balakrishnan


Spectroscopic identification and characterization of covalent and non-covalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. While nuclear magnetic resonance (NMR) methods which provide detailed chemical insights have been successfully employed previously, limited examples are available in the literature for large enzyme complexes. Enzymes utilizing cofactors provide promising examples for such studies when synthetic routes to labeled cofactor analogs and protocols for reconstitution of apo-enzymes with such analogs are readily available. Syntheses of key isotope enriched thiamin diphosphate (ThDP) analogs – [C2, C6 ’ – 13C2] ThDP, [N4 ’ – 15N]ThDP and [C2 – 13C]ThDP – enabled first detection of (i) various ionization/tautomerization states of ThDP during the catalytic cycle of three ThDP dependent enzymes using cross polarization magic angle spinning (CPMAS) solid state NMR (SSNMR) spectroscopy and (ii) [C2, C6 ’ – 13C2] ThDP covalent intermediates on the E1 component (E1p) during the catalytic cycle of E. coli pyruvate dehydrogenase multi-enzyme complex (PDHc) by filter experiments including solution 1-D 1H-13C HSQC NMR. ii

Year: 2016
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