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The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP). The pre-steady state and steady state methods were used to show that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond.

The evidence for the N-terminal region of the PDHc-E1 subunit from E. coli playing a major role in the interaction with the PDHc-E2 subunit is presented. It is concluded that PDHc-E2 protects N-terminal residues 1-55 of PDHc-E1 selectively from trypsin digestion, notwithstanding the fact that the sequence spanning residues give no interpretable electron density in the high-resolution X-ray structure of E.