TitleThiamin diphosphate catalysis in Escherichia coli pyruvate dehydrogenase multi-enzyme complex
NameBalakrishnan, Anand (author), Jordan, Frank (chair), Huskey, Phil (internal member), Hansen, Darren (internal member), Farinas, Edgardo (outside member), Rutgers University, Graduate School - Newark,
Thiamin pyrophosphate-dependent enzymes
DescriptionSpectroscopic 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. Direct evidence was gathered for the 4’-aminopyrimidinium form (APH+) on ThDP molecules bound to (i) S. cerevisiae yeast pyruvate decarboxylase (YPDC) (ii) E1p and (iii) the E1 component of E. coli 2-oxoglutarate dehydrogenase complex (E1o) using 13C and 15N CPMAS SSNMR. The thiazolium C2-H bond was found to be slightly acidic in the cofactor bound to these enzymes. 15N SSNMR experiments confirmed the formation of the 1’,4’-iminopyrimidine tautomer in presence of substrate analogs; a mechanism is proposed for the stabilization of this biologically rare tautomer in enzyme active-sites. Using rapid chemical quench in conjunction with solution NMR, pre-steady state analyses were performed on the native PDHc and PDH complexes reconstituted with E1p active-site loop variants of very low PDHc activity. The C2-α-lactylThDP intermediate could not be detected under any of the conditions used, indicating that its formation is slower than its decarboxylation. The enamine intermediate accumulates at a rate 110 s-1 on E1p and PDHc, while the rates are 100-fold slower for the PDHc variants. 2-acetylThDP could be detected on E1p only during its reaction with pyruvate and the artificial electron acceptor DCPIP. Reductive acetylation of the lipoyl domain in a pre-steady state single turn-over experiment (a model for the E1p-E2p reductive acetyl transfer reaction) was determined by mass spectrometry. Combined, these kinetic results from artificial oxidation reactions suggest the enamine is very well stabilized by E1p and oxidation of the enamine and substrate channeling to E2p are favored by intact PDHc. These studies provide unprecedented insight into the acid-base and covalent electrophilic roles of ThDP in enzyme catalysis and the methods described herein are applicable to all such complexes.
NoteIncludes bibliographical references
Noteby Anand Balakrishnan
CollectionGraduate School - Newark Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.