Pre‐steady State Kinetic Analysis of Covalent Intermediates on the E. coli Pyruvate Dehydrogenase Complex

Abstract

Synthesis of [C2, C6′‐ 13C2]thiamin diphosphate (ThDP) enabled first detection by 1‐D 1H‐13C HSQC NMR of ThDP‐derived intermediates on the E1 component of the pyruvate dehydrogenase complex (PDHc) from E. coli, both in E1 and in the assembled complex. Using rapid chemical quench, pre‐steady state analyses were performed on the PDHc and on its E401K‐E1 and H407A‐E1 variants. The enamine intermediate (detected after acid quench as C2‐alphahydroxyethylThDP) forms at a rate of 80 s‐1 on both the E1 component and on PDHc, while the rates for both are > 100x slower for the E401K‐E1 and H407A‐E1 PDHc variants. Assembly to complex increases the rate of enamine formation for the E401K‐E1 variant ten‐fold. Formation of 2‐acetylThDP could be detected on the E1 component during its reaction with pyruvate and the artificial electron acceptor DCPIP. The rate of formation of this intermediate parallels that of the enamine, probably because protonation of the enamine on the enzyme is much slower than its oxidation. Apparently, the enamine is very well stabilized by the enzyme. On the E401K‐E1 variant 2‐acetylThDP could not be detected, in this case, protonation of the enamine being faster. The C2‐alphalactylThDP intermediate could not be detected under any of the conditions used, indicating that its formation slower than its decarboxylation. The method developed is applicable to all such complexes. Supported by NIH‐GM050380.